Method for producing a mammalian G protein coupled glutamate receptor

ABSTRACT

Mammalian G protein coupled glutamate receptors are identified, isolated and purified. The receptors have been cloned, sequenced and expressed by recombinant means. The receptors and antibodies thereby may be used to identify agonists and antagonists of G protein coupled glutamate receptor mediated neuronal excitation, as well as in methods of diagnosis.

This invention was made with government support under grant number R37AR-17803 awarded by the National Institutes of Health. The governmenthas certain rights in this invention.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.07/648,481, filed Jan. 30, 1991, now abandoned, which is acontinuation-in-part of U.S. patent application Ser. No. 07/626,806,filed Dec. 12, 1990, now abandoned.

BACKGROUND OF THE INVENTION

The majority of nerve cell connections are chemical synapses. Aneurotransmitter is released from the presynaptic terminal, typically inresponse to the arrival of an action potential in the neuron, anddiffuses across the synaptic space to bind to membrane receptor proteinsof the postsynaptic terminal. The binding of neurotransmitters tomembrane receptors is coupled either to the generation of a permeabilitychange in the postsynaptic cell or to metabolic changes.

Neurotransmitters produce different effects according to the type ofreceptor to which they bind. In general, those which produce effectsthat are rapid in onset and brief in duration bind receptors that act asligand-gated ion channels, where binding almost instantly causes an ionflow across the membrane of the postsynaptic cell. Thoseneurotransmitters which act more like local chemical mediators bind toreceptors that are coupled to intracellular enzymes, thereby producingeffects that are slow in onset and more prolonged. Theseneurotransmitters alter the concentration of intracellular secondmessengers in the postsynaptic cell.

Four second messenger systems have been linked to neurotransmitter orhormone receptors and have been studied for their roles in the controlof neuronal excitability. They are the adenylate cyclase/cyclicAMP-dependent protein kinase system, guanylate cyclase andcGMP-dependent protein kinase, the inositol trisphosphate/diacylglycerol-protein kinase C system, and systems which are activated bycalcium ions, such as the calcium/calmodulin-dependent protein kinasesystem. Thus, binding of a transmitter to a receptor may activate, forexample, adenylate cyclase, thereby increasing the intracellularconcentration of cAMP, which in turn activates protein kinases thatphosphorylate specific proteins in the cells, such as those which formion channels and thus alter the cells' electrical behavior. As with theligand-gated ion channel transmitters, the effects can be eitherexcitatory or inhibitory, and may affect the cell at many levels,including the pattern of gene expression. It is also believed that thesechemical synapses, associated with second-messenger systems, may beinvolved in long-term changes that comprise the cellular basis oflearning and memory.

The ligand-activated membrane receptors do not activate the secondmessenger systems directly, however, but via a membrane-bound protein,the GTP-binding protein (G protein), which binds GTP on the cytoplasmicsurface of the cell membrane and thereby acts to couple adenylatecyclase to the membrane receptor. Neurotransmitter binding to themembrane receptor is believed to alter the conformation of the receptorprotein to enable it to activate the G protein in the lipid bilayer,which then binds GTP at the cytoplasmic surface and produces a furtherchange in the G protein to allow it to activate, e.g., an adenylatecyclase molecule to synthesize cAMP. When a ligand binds a receptor, anenzymatic cascade results as each receptor activates several moleculesof G protein, which in turn activate more molecules of adenylate cyclasewhich convert an even larger number of ATPs to cAMP molecules, producinga substantial amplification from the initial event.

Glutamate, aspartate and their endogenous derivatives are believed to bethe predominant excitatory neurotransmitters in the vertebrate centralnervous system. (Krinjrvic, Phys. Rev. 54:418-540, 1974). Recently,glutamate has been described as playing a major, widespread role in thecontrol of neuroendocrine neurons, possibly controlling not only theneuroendocrine system but other hypothalamic regions as well. Four majorsubclasses of glutamate receptors have been described but theircharacterization has until recently been limited to pharmacological andelectrophysiological functional analyses. See generally, Hollman et al.,Nature 342:643-648 (1989) and Sommer et al., Science 249:1580-1585(1990). Three of the receptors, the quisqualate (QA/AMPA), kainate (KA),and N-methyl-D-aspartate (NMDA) receptors, are believed to be directlycoupled to cation-specific ion channels and thus are classified asligand-gated ionotropic receptors. The fourth glutamate receptor bindssome of the agonists of the ionotropic receptors (quisqualate andglutamate, but not AMPA) but has no shared antagonists, and is coupledto G protein. Thus, this receptor, referred to as the G protein-coupledglutamate receptor, or Glu_(G) R, is pharmacologically and functionallydistinct from the other major glutamate receptors. This receptor hasalso been termed the metabotropic receptor.

Agonist binding to Glu_(G) R has been shown to result in the activationof a number of second messenger systems, depending on the systemstudied. One of the best characterized is the quisqualate activation ofphospholipase C through a G protein coupled interaction that leads tothe stimulation of inositol phospholipid metabolism. This activity hasbeen studied in systems that measure the accumulation of radiolabeledinositol monophosphate in response to stimulation by glutamate. Thesystems typically use brain slices from regions such as the hippocampus,striatum, cerebral cortex and hypothalamus (Nicoletti, et al., Proc.Natl. Acad. Sci. USA 83:1931-1935 (1986), and Nicoletti, et al., J.Neurochem. 46:40-46 (1986)) neuronal cultures derived from embryonicmouse and rat cerebellum, corpus striatum and cerebral cortex (Nicolettiet al., J. Neurosci. 6:1905-1911 (1986), Sladeczek et al., Nature317:717-719 (1985), Dumui, et al., Nature 347:182-184 (1990), and Drejeret al., J. Neurosci. 7:2910-2916 (1987)) and rat brain synaptosomes(Recasens et al., Eur. J. Pharm. 141: 87-93 (1987), and Recasens et al.,Neurochem. Int. 13:463-467 (1988)). A major disadvantage of each ofthese model systems is the difficulty in analyzing the pharmacologicaland functional activities of Glu_(G) R in an environment where otherglutamate receptors and G protein-coupled receptors such as muscarinicand serotonin receptors are also present.

The Xenopus oocyte system has been used to identify Glu_(G) R as amember of the family of G protein-coupled receptors. An endogenousinositol triphosphate second messenger-mediated pathway in the oocyteallows the detection of Glu_(G) R after injection of total rat brainmRNA, in that the oocyte responds to ligand via the oocyte Gprotein-coupled PLC-mediated activation of a chloride channel that canbe detected as a delayed, oscillatory current by voltage-clamp recording(Houamed et al., Nature 310:318-321 (1984); Gunderson et al., Proc.Royal Soc. B221:127-143 (1984); Dascal et al., Mol. Brain Res. 1:301-309(1986); Verdoorn et al., Science 238:1114-1116 (1987); Sugiyama et al.,Nature 325:531-533 (1987); Hirono et al., Neuros. Res. 6:106-114 (1988);Verdoorn and Dingledine, Mol. Pharmacol. 34:298-307 (1988); and Sugiyamaet al., Neuron 3:129-132 (1989)). Injection of region-specific brainmRNA and of size fractionated mRNA have suggested that Glu_(G) R may bea large mRNA (6-7 kb) and that it is enriched in the cerebellum (Fong etal., Synapse 2:657-665 (1988) and Horikoshi et al., Neurosci, Lett.105:340-343 (1989)).

There remains considerable need in the art for isolated and purifiedGlu_(G) R, as well as systems capable of expressing Glu_(G) R separatefrom other neurotransmitter receptors. Further, it would be desirable tospecifically identify the presence of Glu_(G) R in cells and tissues,thereby avoiding the time-consuming, complex and nonspecific functionalelectrophysiological and pharmacological assays. It would also bedesirable to screen and develop new agonists and/or antagonists specificfor Glu_(G) R, but to date this has not been practical. Quitesurprisingly, the present invention fulfills these and other relatedneeds.

SUMMARY OF THE INVENTION

The present invention provides isolated and substantially purepreparations of mammalian G protein-coupled glutamate receptors andfragments thereof. In preferred embodiments the receptors are coupled toa G protein in vertebrate cells, bind glutamate and quisqualate andthereby activate phospholipase C, and are capable of stimulatinginositol phospholipid metabolism. Having provided such receptors inisolated and purified form, the invention also provides antibodies tothe receptor, in the form of antisera and/or monoclonal antibodies.

In another aspect the invention provides the ability to produce themammalian G protein-coupled glutamate receptor and polypeptides orfragments thereof by recombinant means, preferably in culturedeukaryotic cells. The expressed receptor or fragments may or may nothave the biological activity of native receptor, and may or may not becoupled to a G protein in the cell used for expression. Accordingly,isolated and purified polynucleotides are described which code for thereceptor and fragments thereof, where the polynucleotides may be in theform of DNA, such as cDNA or RNA. Based on these sequences probes may beused to hybridize and identify these and related genes which encodemammalian G protein-coupled glutamate receptors. The probes may be fulllength cDNA or as small as from 14 to 25 nucleotide, more often thoughfrom about 40 to about 50 or more nucleotides.

In related embodiments the invention concerns DNA constructs whichcomprise a transcriptional promoter, a DNA sequence which encodes thereceptor or fragment, and a transcriptional terminator, each operablylinked for expression of the receptor. For expression the construct mayalso contain at least one signal sequence. The constructs are preferablyused to transform or transfect eukaryotic cells, more preferablymammalian cells which do not express endogenous G protein-coupledglutamate receptors. When bound by an appropriate ligand such asglutamate or quisqualate, the receptor may activate phospholipase C inthe host cell via coupling to G protein. Further, for large scaleproduction the expressed receptor may also be isolated from the cellsby, for example, immunoaffinity purification.

Cells which express the G protein-coupled glutamate receptors may alsobe used to identify compounds which can alter the receptor-mediatedmetabolism of a eukaryotic cell. Compounds may be screened for bindingto the receptor, and/or for effecting a change in receptor-mediatedmetabolism in the host cell. Agonists and/or antagonists of the Gprotein-coupled glutamate receptors may also be screened in cell-freesystems using purified receptors or binding fragments thereof for theeffect on ligand-receptor interaction, or using reconstituted systemssuch micelles which also provide the ability to assess metabolicchanges.

In yet other embodiments the invention relates to methods for diagnosis,where the presence of a mammalian G protein-coupled glutamate receptorin a biological sample may be determined. For example, a monospecificantibody which specifically binds the receptor is incubated with thesample under conditions conducive to immune complex formation, whichcomplexes are then detected, typically by means of a label such as anenzyme, fluorophore, radionuclide, chemiluminiscer, particle, or asecond labeled antibody. Thus, means are provided forimmunohistochemical staining of tissues, including brain tissues, forthe subject receptors.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-C illustrates the construction of plasmid pVEGT, where FIG. 1Ashows the construction of pVEG, FIG. 1B shows the construction of pVEG'and FIG. 1C shows pVEGT'. Symbols used are T7 pro, the T7 promoter; T1and T2, synthetic and native T7 terminators, respectively; M13, M13intergenic region; the parentheses indicate a restriction site destroyedin vector construction; and pA is the Aspergillus niger polyadenylatesequence.

FIG. 2 illustrates representative responses from voltage-clamp assays ofoocytes injected with RNA from positive pools.

FIG. 3 illustrates a partial restriction map of clone 45-A.

FIG. 4 illustrates the cloning of the receptor cDNA present in clone45-A into Zem228R.

FIGS. 5A-H collectively illustrate the DNA sequence and deduced aminoacid sequence of clone 45-A (corresponding to Sequence ID Nos. 1 and 2).Numbers below the line refer to amino acid sequence, numbers to theright of the line refer to nucleotide number. Putative transmembranedomains have been overlined, and putative N-linked glycosylation sitesare indicated by closed circles.

FIG. 6 illustrates a representative dose response curve for varyingconcentrations of L-glutamic acid. Error bars, where larger that thesymbols represent SEM.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Glu_(G) R is a G protein coupled membrane receptor for theneurotransmitter glutamate. As glutamate has been described as having amajor role in the control of neurons, particularly neuroendocrineneurons, Glu_(G) R may play a critical role in effectuating suchcontrol. Consequently, the development of agonists and antagonists ofthe Glu_(G) R-ligand interaction and Glu_(G) R-mediated metabolism is ofgreat interest.

The present invention presents the means to identify agonists andantagonists of the Glu_(G) R-ligand interaction by providing isolatedGlu_(G) R. The term "Glu_(G) R" refers to any protein either derivedfrom a naturally occurring Glu_(G) R, or which shares significantstructural and functional characteristics peculiar to a naturallyoccurring Glu_(G) R. Such a receptor may result when regions of anaturally occurring receptor are deleted or replaced in such a manner asto yield a protein having a similar function. Homologous sequences,allelic variations, and natural mutants; induced point, deletion, andinsertion mutants; alternatively expressed variants; proteins encoded byDNA which hybridize under high or low stringency conditions to nucleicacids which encode naturally occurring Glu_(G) R; proteins retrievedfrom naturally occurring materials; and closely related proteinsretrieved by antisera directed against Glu_(G) R proteins are alsoincluded.

By Glu_(G) R "ligand" is meant a molecule capable of being bound by theligand-binding domain of Glu_(G) R, a Glu_(G) R analog, or chimericGlu_(G) R as generally described in U.S. Pat. No. 4,859,609,incorporated by reference herein. The molecule may be chemicallysynthesized or may occur in nature. Ligands may be grouped into agonistsand antagonists. Agonists are those molecules whose binding to areceptor induces the response pathway within a cell. Antagonists arethose molecules whose binding to a receptor blocks the response pathwaywithin a cell.

By "isolated" Glu_(G) R is meant to refer to Glu_(G) R which is in otherthan its native environment such as a neuron, including, for example,substantially pure Glu_(G) R as defined hereinbelow. More generally,isolated is meant to include Glu_(G) R as a heterologous component of acell or other system. For example, Glu_(G) R may be expressed by a celltransfected with a DNA construct which encodes Glu_(G) R, separated fromthe cell and added to micelles which contain other selected receptors.In another example described below, Glu_(G) R is expressed by a cellwhich has been co-transfected with a gene encoding muscarinic receptor.Thus, in this context, the environment of isolated Glu_(G) R is not asit occurs in its native state, particularly when it is present in asystem as an exogenous component.

The invention provides cloned Glu_(G) R coding sequences which arecapable of expressing the Glu_(G) R protein. Complementary DNA encodingGlu_(G) R may be obtained by constructing a cDNA library from mRNA from,for example, brain tissue. The library may be screened by transcribingthe library and injecting the resulting mRNA into oocytes and detecting,by functional assays, those oocytes which express the Glu_(G) R.Alternatively, the clones may be screened with a complementary labeledoligonucleotide probe.

The present invention relates to successfully isolating a CDNA encodinga Glu_(G) R. Functional cloning of Glu_(G) R was accomplished bysubstantial modifications and improvements to a number of cDNA cloningand molecular biology techniques. Initially, an enriched source ofGlu_(G) R mRNA prepared by sucrose gradient centrifugation of >4 kblength rat cerebellum poly (A)+mRNA was used as template for cDNAsynthesis. Further, a cDNA cloning vector that was employed included apoly (A) tail, thereby increasing by 40-fold the translationalefficiency of the transcription product of the cDNA insert and apolylinker site to allow the directional cloning of the cDNA into thevector between the promoter and the poly (A) tail. Vector constructionfor directional cloning is described in co-pending U.S. Ser. No.07/320,191, incorporated herein by reference. The cDNA cloning vectoralso was used with two transcriptional terminators, in tandem, followingthe poly (A) sequences, efficiently generating a unit length transcriptproduct without non-coding plasmid or viral sequences, and withoutrequiring a restriction endonuclease to linearize the DNA template (astandard practice that will often prevent functional cloning strategiesfrom working due to the presence of the endonuclease site within thecoding region of the cDNA). The cDNA synthesis strategy maximized insertsize and recreation of the 5' end of the cDNA's, without introduction ofhomopolymer tails. cDNA inserts were size-selected to be greater than 4kb in length before insertion into the vector. A library of 10⁶ cDNAinserts in pools of 100,000 were replica plated to cut down on thenumber of amplification steps in the fractionation of sequentiallysmaller pools. Moreover, ml muscarinic cDNA (another G protein-coupledreceptor coupled to phosphoinositol metabolism) template was included intranscription reactions of the subfractionated pools so that beforeinjection the in vitro transcripts from each pool could be assayed byNorthern analysis to assess relative quantity and quality of the mRNA,and by voltage-clamp of oocytes as an internal positive control for eachoocyte not responding to quisqualate or glutamate. The inclusion of adilution of SEAP-VEGT' (a secreted form of alkaline phosphatase)template in transcriptions was also employed so that oocytes selectedfor voltage-clamp analysis were those synthesizing higher levels of theco-injected Glu_(G) R mRNA. And further, low noise electrical recordingtechniques were used to monitor the small signals initially generatedfrom rare transcripts.

With the Glu_(G) R and cDNA clones thereof provided herein, nucleotideand amino acid sequences may be determined by conventional means, suchas by dideoxy sequencing. See generally, Sambrook et al., MolecularCloning, A Laboratory Manual, 2d ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989, incorporated by reference herein.Genomic or cDNA sequences encoding Glu_(G) R and homologous receptors ofthis family may be obtained from libraries prepared from other mammalianspecies according to well known procedures. For instance, usingoligonucleotide probes from rodent Glu_(G) R, such as whole length cDNAor shorter probes of at least about fourteen nucleotides to twenty-fiveor more nucleotides in length; often as many as 40 to 50 nucleotides,DNA sequences encoding Glu_(G) R of other mammalian species, such asiagomorph, avian, bovine, porcine, murine, etc. may be obtained. Ifpartial clones are obtained, it is necessary to join them in properreading frame to produce a full length clone, using such techniques asendonuclease cleavage, ligation and loopout mutagenesis.

A DNA sequence encoding Glu_(G) R is inserted into a suitable expressionvector, which in turn is used to transfect eukaryotic cells. Expressionvectors for use in carrying out the present invention will comprise apromoter capable of directing the transcription of a cloned DNA and atranscriptional terminator.

To direct proteins of the present invention for transport to the plasmamembrane, at least one signal sequence is operably linked to the DNAsequence of interest. The signal sequence may be derived from theGlu_(G) R coding sequence, from other signal sequences described in theart, or synthesized de novo.

Host cells for use in practicing the present invention includemammalian, avian, plant, insect and fungal cells, but preferablymammalian cells. Fungal cells, including species of yeast (e.g.,Saccharomyces spp., particularly S. cerevisiae, Schizosaccharomycesspp.) or filamentous fungi (e.g., Aspergillus spp., Neurospora spp.) maybe used as host cells within the present invention. Suitable yeastvectors for use in the present invention include YRp7 (Struhl et al.,Proc. Natl. Acad. Sci. USA. 76: 1035-1039, 1978), YEp13 (Broach et al.,Gene 8: 121-133, 1979), POT vectors (Kawasaki et al, U.S. Pat. No.4,931,373, which is incorporated by reference herein), pJDB249 andpJDB219 (Beggs, Nature 275:104-108, 1978) and derivatives thereof. Suchvectors will generally include a selectable marker, which may be one ofany number of genes that exhibit a dominant phenotype for which aphenotypic assay exists to enable transformants to be selected.Preferred selectable markers are those that complement host cellauxotrophy, provide antibiotic resistance or enable a cell to utilizespecific carbon sources, and include LEU2 (Broach et al., ibid.), URA3(Botstein et al., Gene 8: 17, 1979), HIS3 (Struhl et al., ibid.) or POT1(Kawasaki et al., ibid.). Another suitable selectable marker is the CATgene, which confers chloramphenicol resistance on yeast cells.

Additional vectors, promoters and terminators for use in expressing thereceptor of the invention in yeast are well known in the art and arereviewed by, for example, Emr, Meth. Enzymol. 185:231-279, (1990),incorporated herein by reference. The receptors of the invention may beexpressed in Aspergillus spp. (McKnight and Upshall, described in U.S.Pat. No. 4,935,349, which is incorporated herein by reference). Usefulpromoters include those derived from Aspergillus nidulans glycolyticgenes, such as the ADH3 promoter (McKnight et al., EMBO J. 4:2093-2099,1985) and the tpiA promoter. An example of a suitable terminator is theADH3 terminator (McKnight et al., ibid.). Techniques for transformingfungi are well known in the literature, and have been described, forinstance by Beggs (ibid.), Hinnen et al. (Proc. Natl. Acad. Sci. USA75:1929-1933, 1978), Yelton et al. (Proc. Natl. Acad. Sci. USA 81:1740-1747, 1984), and Russell (Nature 301:167-169, 1983) each of which areincorporated herein by reference.

A variety of higher eukaryotic cells may serve as host cells forexpression of the Glu_(G) R, although not all cell lines will be capableof functional coupling of the receptor to the cell's second messengersystems. Cultured mammalian cells, such as BHK, CHO, Y1 (Shapiro et al.,TIPS Suppl. 43-46 (1989)), NG108-15 (Dawson et al., NeuroscienceApproached Through Cell Culture, Vol. 2, pages 89-114 (1989)), N1E-115(Liles et al., J. Biol. Chem. 261:5307-5313 (1986)), PC 12 and COS-1(ATCC CRL 1650) are preferred. Preferred BHK cell lines are the tk⁻⁻ts13 BHK cell line (Waechter and Baserga, Proc. Natl. Acad. Sci. USA79:1106-1110 (1982)) and the BHK 570 cell line (deposited with theAmerican Type Culture Collection, 12301 Parklawn Dr., Rockville, Md.under accession number CRL 10314). A tk⁻⁻ BHK cell line is availablefrom the ATCC under accession number CRL 1632.

Mammalian expression vectors for use in carrying out the presentinvention will include a promoter capable of directing the transcriptionof a cloned gene or cDNA. Preferred promoters include viral promotersand cellular promoters. Viral promoters include the immediate earlycytomegalovirus promoter (Boshart et al., Cell 41: 521-530, 1985) andthe SV40 promoter (Subramani et al., Mol. Cell. Biol. 1: 854-864, 1981).Cellular promoters include the mouse metallothionein-1 promoter(Palmiter et al., U.S. Pat. No. 4,579,821), a mouse V_(k) promoter(Bergman et al., Proc. Natl. Acad. Sci. USA 81: 7041-7045, 1983; Grantet al., Nuc. Acids Res. 15: 5496, 1987) and a mouse V_(H) promoter (Lohet al., Cell 33: 85-93, 1983). A particularly preferred promoter is themajor late promoter from Adenovirus 2 (Kaufman and Sharp, Mol. Cell.Biol. 2: 1304-13199, 1982). Such expression vectors may also contain aset of RNA splice sites located downstream from the promoter andupstream from the DNA sequence encoding the peptide or protein ofinterest. Preferred RNA splice sites may be obtained from adenovirusand/or immunoglobulin genes.

Also contained in the expression vectors is a polyadenylation signallocated downstream of the coding sequence of interest. Polyadenylationsignals include the early or late polyadenylation signals from SV40(Kaufman and Sharp, ibid.), the polyadenylation signal from theAdenovirus 5 E1B region and the human growth hormone gene terminator(DeNoto et al., Nuc. Acid Res. 9: 3719-3730, 1981). The expressionvectors may include a noncoding viral leader sequence, such as theAdenovirus 2 tripartite leader, located between the promoter and the RNAsplice sites. Preferred vectors may also include enhancer sequences,such as the SV40 enhancer and the mouse μ enhancer (Gillies, Cell 33:717-728, 1983). Expression vectors may also include sequences encodingthe adenovirus VA RNAs.

Cloned DNA sequences may be introduced into cultured mammalian cells by,for example, calcium phosphate-mediated transfection (Wigler et al.,Cell 14: 725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7: 603,1981; Graham and Van der Eb, Virology 52: 456, 1973.) Other techniquesfor introducing cloned DNA sequences into mammalian cells, such aselectroporation (Neumann et al., EMBO J. 1: 841-845, 1982), may also beused. In order to identify cells that have integrated the cloned DNA, aselectable marker is generally introduced into the cells along with thegene or cDNA of interest. Preferred selectable markers for use incultured mammalian cells include genes that confer resistance to drugs,such as neomycin, hygromycin, and methotrexate. The selectable markermay be an amplifiable selectable marker. Preferred amplifiableselectable markers are the DHFR gene and the neomycin resistance gene.Selectable markers are reviewed by Thilly (Mammalian Cell Technology,Butterworth Publishers, Stoneham, MA, which is incorporated herein byreference). The choice of selectable markers is well within the level ofordinary skill in the art.

Selectable markers may be introduced into the cell on a separate plasmidat the same time as the gene of interest, or they may be introduced onthe same plasmid. If on the same plasmid, the selectable marker and thegene of interest may be under the control of different promoters or thesame promoter, the latter arrangement producing a dicistronic message.Constructs of this type are known in the art (for example, Levinson andSimonsen, U.S. Pat. No. 4,713,339). It may also be advantageous to addadditional DNA, known as "carrier DNA" to the mixture which isintroduced into the cells.

Transfected mammalian cells are allowed to grow for a period of time,typically 1-2 days, to begin expressing the DNA sequence(s) of interest.Drug selection is then applied to select for growth of cells that areexpressing the selectable marker in a stable fashion. Transfected cellsmay also be selected in the presence of antagonist to inhibit theactivity of the receptor. Suitable antagonists in this context includeD, L, 2-amino-3-phosphonopropionate. For cells that have beentransfected with an amplifiable selectable marker the drug concentrationmay be increased in a stepwise manner to select for increased copynumber of the cloned sequences, thereby increasing expression levels.

Promoters, terminators and methods suitable for introducing expressionvectors encoding recombinant Glu_(G) R into plant, avian and insectcells are known in the art. The use of baculoviruses, for example, asvectors for expressing heterologous DNA sequences in insect cells hasbeen reviewed by Atkinson et al. (Pestic. Sci. 28: 215-224,1990). Theuse of Agrobacterium rhizogenes as vectors for expressing genes in plantcells has been reviewed by Sinkar et al. (J. Biosci. (Banglaore) 11:47-58, 1987).

Host cells containing DNA constructs of the present invention are thencultured to produce recombinant Glu_(G) R. The cells are culturedaccording to accepted methods in a culture medium containing nutrientsrequired for growth of mammalian or other host cells. A variety ofsuitable media are known in the art and generally include a carbonsource, a nitrogen source, essential amino acids, vitamins, minerals andgrowth factors. The growth medium will generally select for cellscontaining the DNA construct by, for example, drug selection ordeficiency in an essential nutrient which is complemented by theselectable marker on the DNA construct or co-transfected with the DNAconstruct.

The Glu_(G) R produced according to the present invention may bepurified from the recombinant expression systems or other sources usingpurification protocols that employ techniques generally available tothose skilled in the art. The most convenient sources for obtaininglarge quantities of Glu_(G) R are cells which express the recombinantreceptor. However, other sources, such as tissues, particularly braintissues of the cerebellum which contain Glu_(G) R may also be employed.

Purification may be achieved by conventional chemical purificationmeans, such as liquid chromatography, lectin affinity chromatography,gradient centrifugation, and gel electrophoresis, among others. Methodsof protein purification are known in the art (see generally, Scopes, R.,Protein Purification, springer-Verlag, N.Y. (1982), which isincorporated herein by reference) and may be applied to the purificationof the Glu_(G) R and particularly the recombinantly produced Glu_(G) Rdescribed herein. In a preferred embodiment immunoaffinitychromatography is employed using antibodies directed against Glu_(G) Ras herein described. In another method of purification, the recombinantgene encoding Glu_(G) R or portions thereof can be modified at the aminoterminus, just behind a signal peptide, with a sequence coding for asmall hydrophilic peptide, such as described in U.S. Pat. Nos. 4,703,004and 4,782,137, incorporated herein by reference. Specific antibodies forthe peptide facilitate rapid purification of Glu_(G) R.sub., and theshort peptide can then be removed with enterokinase.

Thus, as discussed above, the present invention provides Glu_(G) Risolated from its natural cellular environment, substantially free ofother G protein coupled glutamate receptors. Purified Glu_(G) R is alsoprovided. Substantially pure Glu_(G) R of at least about 50% ispreferred, at least about 70-80% more preferred, and 95-99% or morehomogeneity most preferred, particularly for pharmaceutical uses. Oncepurified, partially or to homogeneity, as desired, the recombinantGlu_(G) R or native Glu_(G) R may then be used to generate antibodies,in assay procedures, etc.

In another aspect, the invention concerns polypeptides and fragments ofGlu_(G) R. Polypeptides and fragments of Glu_(G) R may be isolated fromrecombinant expression systems or may be synthesized by the solid phasemethod of Merrifield, Fed. Proc. 21:412 (1962), Merrifield, J. Am. Chem.Soc. 85:2149 (1963), or Barany and Merrifield, in The Peptides, vol. 2,pp. 1-284 (1979) Academic Press, N.Y., each of which are incorporatedherein by reference, or by use of an automated peptide synthesizer. By"polypeptides" is meant a sequence of at least about 3 amino acids,typically 6 or more, up to 100-200 amino acids or more, including entireproteins. For example, the portion(s) of Glu_(G) R protein which bindsligand may be identified by a variety of methods, such as by treatingpurified receptor with a protease or a chemical agent to fragment it anddetermine which fragment is able to bind to labeled glutamate in aligand blot. Polypeptides may then be synthesized and used as antigen,to inhibit ligand-Glu_(G) R interaction, etc. It should be understoodthat as used herein, reference to Glu_(G) R is meant to include theprotein, polypeptides, and fragments thereof unless the contextindicates otherwise.

In another aspect, the invention provides means for regulating theGlu_(G) R-ligand interaction, and thus treating, therapeutically and/orprophylactically, a disorder which can be linked directly or indirectlyto Glu_(G) R or to its ligands, such as glutamate and other endogenousexcitatory amino acids. By virtue of having the receptor of theinvention, agonists or antagonists may be identified which stimulate orinhibit the interaction of ligand with Glu_(G) R. With either agonistsor antagonists the metabolism and reactivity of cells which express thereceptor are controlled, thereby providing a means to abate or in someinstances prevent the disease of interest.

Thus, the invention provides screening procedures for identifyingagonists or antagonists of events mediated by the ligand-Glu_(G) Rinteraction. Such screening assays may employ a wide variety of formats,depending to some extent on which aspect of the ligand/receptor/Gprotein interaction is targeted. For example, such assays may bedesigned to identify compounds which bind to the receptor and therebyblock or inhibit interaction of the receptor with the ligand. Otherassays can be designed to identify compounds which can substitute forligand and therefore stimulate Glu_(G) R-mediated intracellularpathways. Yet other assays can be used to identify compounds whichinhibit or facilitate the association of Glu_(G) R to G protein andthereby mediate the cellular response to Glu_(G) R ligand.

In one functional screening assay, the initiation of fertilizationactivation events are monitored in eggs which have been injected with,e.g., mRNA which codes for Glu_(G) R and subsequently exposed toselected compounds which are being screened, in conjunction with orapart from an appropriate ligand. See generally, Kline et al., Science241:464-467 (1988), incorporated herein by reference.

Another screening assay is based on the use of mammalian cell lineswhich express Glu_(G) R functionally coupled to a mammalian G protein.In this assay, compounds are screened for their relative affinity asreceptor agonists or antagonists by comparing the relative receptoroccupancy to the extent of ligand induced stimulation or inhibition ofsecond messenger metabolism. For example, activation of phospholipase Cleads to increased inositol monophosphate metabolism. Means formeasuring inositol monophosphate metabolism are generally described inSubers and Nathanson, J. Mol. Cell, Cardiol. 20:131-140 (1988),incorporated herein by reference.

The screening procedure can be used to identify reagents such asantibodies which specifically bind to the receptor and substantiallyaffect its interaction with ligand, for example. The antibodies may bemonoclonal or polyclonal, in the form of antiserum or monospecificantibodies, such as purified antiserum or monoclonal antibodies ormixtures thereof. For administration to humans, e.g., as a component ofa composition for in yivo diagnosis or imaging, the antibodies arepreferably substantially human to minimize immunogenicity and are insubstantially pure form. By substantially human is meant generallycontaining at least about 70% human antibody sequence, preferably atleast about 80% human, and most preferably at least about 90-95% or moreof a human antibody sequence to minimize immunogenicity in humans.

Antibodies which bind Glu_(G) R may be produced by a variety of means.The production of non-human antisera or monoclonal antibodies, e.g.,murine, lagomorpha, equine, etc. is well known and may be accomplishedby, for example, immunizing the animal with the receptor molecule or apreparation containing a desired portion of the receptor molecule, suchas that domain or domains which contributes to ligand binding. For theproduction of monoclonal antibodies, antibody producing cells obtainedfrom immunized animals are immortalized and screened, or screened firstfor the production of antibody which binds to the receptor protein andthen immortalized. As the generation of human monoclonal antibodies tohuman Glu_(G) R antigen may be difficult with conventional techniques,it may be desirable to transfer antigen binding regions of the non-humanantibodies, e.g. the F(ab')₂ or hypervariable regions, to human constantregions (Fc) or framework regions by recombinant DNA techniques toproduce substantially human molecules. Such methods are generally knownin the art and are described in, for example, U.S. Pat. No. 4,816,397,EP publications 173,494 and 239,400, which are incorporated herein byreference. Alternatively, one may isolate DNA sequences which code for ahuman monoclonal antibody or portions thereof that specifically bind tothe human receptor protein by screening a DNA library from human B cellsaccording to the general protocol outlined by Huse et al., Science246:1275-1281 (1989), incorporated herein by reference, and then cloningand amplifying the sequences which encode the antibody (or bindingfragment) of the desired specificity.

In other embodiments, the invention provides screening assays conductedin vitro with cells which express the receptor. For example, the DNAwhich encodes the receptor or selected portions thereof may betransfected into an established cell line, e.g., a mammalian cell linesuch as BHK or CHO, using procedures known in the art (see, e.g.,Sambrook et al., Molecular Cloning, A Laboratory Manual, 2d ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, which isincorporated herein by reference). The receptor is then expressed by thecultured cells, and selected agents are screened for the desired effecton the cell, separately or in conjunction with an appropriate ligandsuch as glutamate or quisqualate. Means for amplifying nucleic acidsequences which may be employed to amplify sequences encoding thereceptor or portions thereof are described in U.S. Pat. Nos. 4,683,195and 4,683,202, incorporated herein by reference.

In yet another aspect, the screening assays provided by the inventionrelate to transgenic mammals whose germ cells and somatic cells containa nucleotide sequence encoding Glu_(G) R protein or a selected portionof the receptor which, e.g., binds ligand, GTP binding protein, or thelike. There are several means by which a sequence encoding, for example,the human Glu_(G) R may be introduced into a non-human mammalian embryo,some of which are described in, e.g., U.S. Pat. No. 4,736,866, Jaenisch,Science 240-1468-1474 (1988) and Westphal et al., Annu. Rev. Cell Biol.5:181-196 (1989), which are incorporated herein by reference. Theanimal's cells then express the receptor and thus may be used as aconvenient model for testing or screening selected agonists orantagonists.

In another aspect the invention concerns diagnostic methods andcompositions. By means of having the Glu_(G) R molecule and antibodiesthereto, a variety of diagnostic assays are provided. For example, withantibodies, including monoclonal antibodies, to Glu_(G) R.sub., thepresence and/or concentration of receptor in selected cells or tissuesin an individual or culture of interest may be determined. These assayscan be used in the diagnosis and/or treatment of diseases such as, forexample, cerebral ischemia, Parkinsons, senile dementia and othercognitive disorders, Huntington's chorea, amyotrophic lateral sclerosis,emesis, migraine, and others.

Numerous types of immunoassays are available and are known to thoseskilled in the art, e.g., competitive assays, sandwich assays, and thelike, as generally described in, e.g., U.S. Pat. Nos. 4,642,285;4,376,110; 4,016,043; 3,879,262; 3,852,157; 3,850,752; 3,839,153;3,791,932; and Harlow and Lane, Antibodies, A Laboratory Manual, ColdSpring Harbor Publications, N.Y. (1988), each incorporated by referenceherein. In one assay format Glu_(G) R is identified and/or quantified byusing labeled antibodies, preferably monoclonal antibodies which arereacted with brain tissues, e.g., cortex, striatum, hippocampus,cerebellum, and determining the specific binding thereto, the assaytypically being performed under conditions conducive to immune complexformation. Unlabeled primary antibody can be used in combination withlabels that are reactive with primary antibody to detect the receptor.For example, the primary antibody may be detected indirectly by alabeled secondary antibody made to specifically detect the primaryantibody. Alternatively, the anti-Glu_(G) R antibody can be directlylabeled. A wide variety of labels may be employed, such asradionuclides, particles (e.g., gold, ferritin, magnetic particles, redblood cells), fluorophores, chemiluminescers, enzymes, enzymesubstrates, enzyme cofactors, enzyme inhibitors, ligands (particularlyhaptens), etc.

The Glu_(G) R DNA may be directly detected in cells with a labeledGlu_(G) R DNA or synthetic oligonucleotide probe in a hybridizationprocedure similar to the Southern or dot blot. Also, the polymerasechain reaction (Saiki et al., Science 239:487 (1988), and U.S. Pat. No.4,683,195) may be used to amplify DNA sequences, which are subsequentlydetected by their characteristic size on agarose gels, Southern blot ofthese gels using Glu_(G) R DNA or a oligonucleotide probe, or a dot blotusing similar probes. The probes may comprise from about 14 nucleotidesto about 25 or more nucleotides, preferably, 40 to 60 nucleotides, andin some instances a substantial portion or even the entire cDNA ofGlu_(G) R may be used. The probes are labeled with a with a detectablesignal, such as an enzyme, biotin, a radionuclide, fluorophore,chemiluminescer, paramagnetic particle, etc.

Kits can also be supplied for use with the receptor of the subjectinvention in the detection of the presence of the receptor or antibodiesthereto, as might be desired in the case of autoimmune disease. Thus,antibodies to Glu_(G) R, preferably monospecific antibodies such asmonoclonal antibodies, or compositions of the receptor may be provided,usually in lyophilized form in a container, either segregated or inconjunction with additional reagents, such as anti-antibodies, labels,gene probes, polymerase chain reaction primers and polymerase, and thelike.

The following examples are offered by way of illustration, not bylimitation.

EXAMPLE I Preparation of Glu_(G) R Enriched mRNA

Total RNA was prepared from the cerebellum of rats using guanidineisothiocyanate (Chirgwin et al. Biochemistry 18:52-94 (1979)) and CsClcentrifugation. Poly (A)+RNA was isolated using oligo d(T) cellulosechromatography. After 2 rounds of chromatography on oligo d(T) cellulosethe RNA (800 μg) was divided into two aliquots and layered over 10-40%linear sucrose gradients in tubes for an SW 28 rotor. The gradients werecentrifuged for 28 hours at 25,000 rpm to pellet RNA greater than 4 kbin size. The enriched RNA was injected into frog oocytes and assayed forthe presence of the Glu_(G) R.

Injection of Oocytes and Voltage-Clamp Assay of Glu_(G) R Activity

Oocytes were prepared from ovarian lobes that were surgically removedfrom anesthetized Xenopus females. The ovarian lobes were washed, pulledapart into small clumps and dissociated by treatment with collagenasefor 2-3 hours at 20° C. with constant, gentle agitation. Thedissociation and defolicularization of the oocytes is completed manuallyafter removal of the collagenase. Oocytes that were judged healthy andgreater than 1 mm in diameter were transferred to a 50 mm sterile tissueculture dish and incubated in sterile, antibiotic-supplemented Barth'smedium (88 mM NaCl, 1 mM KCl, 0.82 mM MgSO₄, 0.33 mM Ca(NO₃)₂, 0.41 mMCaCl₂, 2.4 mM NaHCO₃, 10 mM HEPES, pH 7.4, 0.1 mg/ml gentamicin, 0.01mg/ml penicillin, 0.01 mg/ml streptomycin, 0.5 mM theophylline, and 2.5mM Na pyruvate) at 19° C.

Injection pipettes were pulled from hard glass tubing (Drummond) on amodified 700C Kopf vertical puller. The tip was broken and bevelledusing a List Medical microforge. Tip diameters of the pipettes rangedfrom 20-30 mM. Injection pipettes were made RNase free by heating to285° C. overnight.

Following overnight incubation, healthy oocytes were selected forinjection. RNA, which was stored at -70° C. in DEPC-treated water, wasthawed and centrifuged at 15,000 g for five minutes. Injection wasperformed using a modified pipetting device (Drummond). After injection,the oocytes were incubated in fresh, sterile Barth's medium which waschanged daily, and unhealthy oocytes were removed.

Voltage-clamp assays were carried out on injected oocytes which wereeach placed in a small chamber of approximately 500 μl in volume andwhich was continuously perfused with standard frog Ringer's (115 mMNaCl, 2.5 mM KC1, 1.8 mM CaCl₂, 10 mM HEPES, pH 7.2) at 1-6 ml/min. Theoocyte was impaled with two glass microelectrodes for recording which,when filled with 3 M KCl, had a tip resistance of 0.5 to 7.0 megaohms.One of the two electrodes was connected to a differential amplifier viaa silver/silver chloride half cell. The bath potential was measured byconnecting the other side of the differential amplifier to the bath viaa silver/silver chloride pellet and a Ringer/Agar bridge. A low noise,high compliance, voltage-clamp system (NPI) was used to control themembrane potential and to measure membrane current. The oocyte membranepotential was maintained at -60 mV (inside cell negative). Onemillimolar glutamate (Sigma), 100 μM quisqualate (Sigma), 1 mMcarbamylcholine (Sigma), and the other drugs used in this assay wereapplied by switching the perfusing medium to a medium containing a drugfor approximately three minutes, and the membrane current was recordedon a chart recorder (Linear Instruments).

After impaling the oocyte with the two microelectrodes, and imposing thevoltage-clamp, the membrane current (the holding current) graduallydeclines to a steady state over a period of several minutes. When theholding current stabilizes, so that the chart record is horizontal, thedrug is applied for one to three minutes. An oocyte is judged to have apositive response if a rapid inward current spike (downward deflectionon the chart), followed by slow current oscillations of decreasingmagnitude, is observed. Our lower limit of detection depended on thesteadiness of the holding current prior to drug application, but was inthe range of 5-10 nA.

Construction of pVEGT'

To permit transcription of cloned cDNA without prior endonucleasedigestion, bacteriophage T7 transcriptional terminators were added to acloning vector. Plasmid pVEGT' is described in copending U.S. Ser. No.07/581,342, which is incorporated by reference herein. The sequence ofthe putative T7 RNA transcription terminator, which lies between gene 10and gene 11 of bacteriophage T7, is disclosed by Dunn and Studier (J.Mol. Biol. 166:477-536 (1983)). As shown in FIG. 5, four syntheticoligonucleotides were designed from this sequence and ligated into thevector pGEM-1 (obtained from Promega Biotec, Madison, Wis.), a plasmidcontaining a bacterial origin of replication, ampicillin resistancegene, and the T7 promoter adjacent to a multiple cloning site. Terminalphosphates were added to the 5' ends of oligonucleotides ZC776 and ZC777(Sequence ID Nos. 4 and 5) with T4 polynucleotide kinase and ATP, understandard conditions (Maniatis et al. ibid). (The sequences of these andother oligonucleotides referred to herein are shown in Table 1.) Afterthe incubation, the kinase was heat killed at 65° C. for 10 min.Twenty-five oligonucleotide ZC775 (Sequence ID Number 3) and 25 ng ofoligonucleotide ZC776 (Sequence ID Number 4) were annealed by incubationat 65° C. for 15 minutes, then allowed to cool to room temperature in500 ml of water. Oligonucleotides ZC777 and ZC778 (Sequence ID Nos. 5and 6) were similarly annealed. The annealed oligonucleotides werestored at -20° C. until use. The vector pGEM-1 was digested with Pst Iand Hind III, and the linearized vector DNA was purified by agarose gelelectrophoresis. The synthetic T7 terminator (annealed oligonucleotidesZC775, ZC776, ZC777 and ZC778; Sequence ID Nos. 3, 4, 5 and 6) was thencloned into pGEM-1. Twenty-five nanograms of vector plus an equal molaramount of each of the annealed oligonucleotides ZC775/ZC776 (Sequence IDNos. 3 and 4) and ZC777/ZC778 (Sequence ID Nos. 5 and 6) were combinedin a 10 μl reaction mix. After an overnight ligation at 14° C., the DNAwas transformed into competent E. coli JM83 cells, and the transformedcells were selected for ampicillin resistance. Plasmid DNA was preparedfrom selected transformants by the alkaline lysis procedure (Birnboimand Doly, Nuc. Acids Res. 7:1513-1523 (1979)). A portion of the DNA fromthese samples was cut with Pst I and Hind III and analyzed on a 4%polyacrylamide gel to identify clones that released an 80 bp Pst I-HindIII fragment. Other diagnostic cuts, such as Eco RI and Not I, were alsomade. One of the isolates, designated pGEMT, was shown by restrictionanalysis to contain the T7 terminator fragment.

                                      TABLE 1                                     __________________________________________________________________________    Oligonucleotide Sequences (5'-3')                                             __________________________________________________________________________    ZC775 (Sequence ID Number 3):                                                 GCT AGC ATA ACC CCT TGG GGC CTC TAA ACG GGT CT                                ZC776 (Sequence ID Number 4):                                                 CTC AAG ACC CGT TTA GAG GCC CCA AGG GGT TAT GCT AGC TGC A                     ZC777 (Sequence ID Number 5):                                                 TGA GGG GTT TTT TGC TGA AAG GAG GAA CTA TGC GGC CGC A                         ZC778 (Sequence ID Number 6):                                                 AGC TTG CGG CCG CAT AGT TCC TCC TTT CAG CAA AAA ACC C                         ZC1751 (Sequence ID Number 7):                                                AAT TCT GTG CTC TGT CAA G                                                     ZC1752 (Sequence ID Number 8):                                                GAT CCT TGA CAG AGC ACA G                                                     ZC2063 (Sequence ID Number 9):                                                GAT CCA AAC TAG TAA AAG AGC T                                                 ZC2064 (Sequence ID Number 10):                                               CTT TTA CTA GTT TG                                                            ZC2938 (Sequence ID Number 11):                                               GAC AGA GCA CAG ATT CAC TAG TGA GCT CTT TTT TTT TTT TTT T                     ZC3015 (Sequence ID Number 12):                                               TTC CAT GGC ACC GTC AAG GCT                                                   ZC3016 (Sequence ID Number 13):                                               AGT GAT GGC ATG GAC TGT GGT                                                   ZC3652 (Sequence ID Number 14):                                               ACA TGC ACC ATG CTC TGT GT                                                    ZC3654 (Sequence ID Number 15):                                               AGT GAT GGC ATG GAC TGT GGT                                                   __________________________________________________________________________

The native T7 terminator from plasmid pAR2529 (Rosenberg et al., Gene56:125-135 (1987)) was added to plasmid pGEMT. Plasmid pGEMT wasdigested with Bam HI and plasmid pAR2529 was digested with Bam HI andBgl II (FIG. 1). The Bam HI-Bgl II terminator fragment from pAR2529 waspurified by agarose gel electrophoresis. The terminator fragment wasligated to Bam HI digested pGEMT, and the DNA was transformed intocompetent E. coli LM1035 cells. Colonies that were ampicillin resistantwere inoculated into 5 ml cultures for overnight growth. Plasmid DNAprepared by the alkaline lysis procedure was screened for properterminator orientation by Bam HI-Sal I digestion and electrophoresis onan 8% polyacrylamide gel. A clone that contained the terminator in thecorrect orientation, as evidenced by the presence of a 130 bp Bam HI-SalI fragment, was chosen and named pGEMTT (FIG. 1).

To allow pGEMTT to be packaged as single-stranded DNA in the presence ofM13 phage proteins, the M13 intergenic region from pUC382 (similar topUC118 and 119 as disclosed by Vieira and Messing, Methods Enzymol. 153:3-11 (1987)) was added to pGEMTT (FIG. 1). Plasmid pGEMTT was digestedwith Fsp I and Nar I, and the fragment containing the T7 promoter andtranscription terminator was purified. Plasmid pUC382 was digested withFsp I and Nar I, and the fragment encoding the ampicillin resistancegene and the M13 intergenic region was gel purified. These fragmentswere then ligated together in the presence of T4 DNA ligase. The ligatedDNA was transformed into competent E. coli LM1035 cells. Plasmid DNAfrom twelve ampicillin-resistant colonies was prepared by the alkalinelysis method, and the DNA was screened by digestion with Ava I. Theappropriate construction gave two bands, one of 2430 bp and another of709 bp. One such isolate was chosen and named pVEG. Syntheticoligonucleotides encoding the prime sequence were added to pVEG betweenthe Bam HI and Eco RI sites (FIG. 1). Plasmid pVEG was digested with BamHI and Eco RI and the vector fragment was gel purified. Ninety-sixnanograms each of oligonucleotides ZC1751 and ZC1752 (Sequence ID Nos. 7and 8) were annealed in 4.5 μl of 10 mM Tris pH 7.5, 20 mM MgCl₂ and 10mM NaCl at 65° C. for 20 minutes, then the mixture was cooled to roomtemperature over a period of 30 minutes. The annealed oligonucleotideswere ligated to the pVEG vector fragment with T4 DNA ligase and thentransformed into competent E. coli LM1035 cells. After growing overnightto develop the colonies, a filter lift was taken of the colonies on theagar plate. The filter was probed with 32P-labeled oligonucleotideZC1751 (Sequence ID Number 7). All of the colonies were positive.Plasmid DNA was prepared from cultures grown from 12 of the colonies.The plasmid DNA was screened by digestion with Sst I to verify theabsence of the Sst I site between the Eco RI and Bam HI sites of pVEG.All 12 of the plasmid DNAs were negative for Sst I digestion. One ofthese 12 isolates was chosen and named pVEG'.

A polyadenylate sequence derived from an Aspergillus alcoholdehydrogenase cDNA was added to pVEG. As shown in FIG. 1, plasmid pM098(disclosed in published European patent application EP 272,277 anddeposited with American Type Culture Collection under accession number53428) was digested with Dra I and Bam HI, and the approximately 150 bppoly(A) fragment was purified by agarose gel electrophoresis. Thisfragment contained mostly poly(A) sequence with very little flankingcDNA. To clone the poly(A) cDNA fragment into pVEG, pVEG was digestedwith Bam HI and Sma I, and the 3.4 kb vector fragment was gel purified.The vector and poly(A) fragments were ligated together with T4 DNAligase to produce vector pVEGT (FIG. 1).

Synthetic oligonucleotides encoding the prime sequence were added topVEGT. To accomplish this, pVEGT was digested with Not I and Sst I, andthe 370 bp fragment containing the poly(A) sequence and the two T7transcriptional terminators was purified by agarose gel electrophoresis.Plasmid pVEG' was digested with Not I and Bam HI, and the 3.2 kb vectorfragment was gel-purified. Two oligonucleotides (ZC2063 and ZC2064;Sequence ID Nos. 9 and 10) that formed, when annealed, a Bam HI-Sst Iadapter were synthesized. The two oligonucleotides were individuallykinased and annealed, and ligated with the linearized vector and thepoly(A)-terminator fragment. The resultant vector, designated pVEGT'(FIG. 1), contained a T7 RNA transcription promoter, an Eco RI cloningsite flanked by the prime sequence, a poly(A) tract, and two T7 RNApolymerase terminators.

Construction of cDNA Library from Rat Cerebellum Poly (A)+RNA

Because there was evidence suggesting that the Glu_(G) R was encoded avery large mRNA of 7 kb (Fong, Davidson, and Lester, Synapse 2:657(1988)) and because full length cDNA encompassing the coding sequence isrequired for functional cloning of cDNA, measures were taken to optimizefor synthesis of large cDNA. A novel method of cDNA synthesis wasdeveloped which yielded large full length cDNA. This was evident bydemonstration that full length 7.5 kb cDNA could be synthesized from amodel 7.5 kb mRNA and that large full length cDNA were present in alibrary constructed from poly (A)+RNA as demonstrated by Southern blotanalysis. In addition, all enzymes which were important in this methodwere pretested and selected from a large number of lots of enzymesavailable from commercial suppliers. Once a satisfactory lot wasidentified, a large amount of the enzyme was purchased and the enzymewas stored at -70° C. until used. Once used, the enzyme was stored at-20° C. for a few months and then discarded. Different "lots" of enzymesfrom commercial suppliers, including lots of Superscript reversetranscriptase (BRL), E. coli DNA polymerase I (Amersham) and Mung beannuclease (NEB), which were used in the cDNA synthesis, were screened forquality in test synthesis assays. Superscript reverse transcriptase lotswere assayed for the ability to synthesize unit length (7.5 kb) firststrand cDNA from 7.5 kb RNA (BRL) control. Conditions for first strandsynthesis with Superscript reverse transcriptase lots were prepared asdescribed below. Radiolabeled first strand cDNA was analyzed by alkalineagarose gel electrophoresis. SUPERSCRIPT reverse transcriptase lotscapable of producing unit length, 7.5 kb cDNA were selected for use.

E. coli DNA polymerase I lots were assayed for the ability to produce,by hairpin DNA formation, full-length second strand cDNA from the 7.5 kbunit-length first strand cDNA. The second strand cDNA syntheses werecarried out as described below. The quality of the second strandsyntheses were assessed by alkaline agarose electrophoresis of theradiolabeled product. DNA polymerase I lots capable of producing 15 kbsecond strand DNA from the 7.5 kb unit length first strand cDNA wereselected for use.

Mung bean nuclease lots were tested for the ability to clip the hairpinDNA formed during second strand synthesis without degrading the cDNA. Inaddition, varying concentrations of enzyme were added to determine theoptimum enzyme concentration for the conditions set forth below. Thereactions were assessed by alkaline agarose electrophoresis. Lots andconcentrations resulting in the production of 7.5 kb unit length cDNAwere selected for use.

Total RNA was prepared from rat cerebella using guanidine isothiocyanate(Chirgwin et al. Biochemistry 18:52-94 1979) and CsCl centrifugation(Gilsin et al. Biochemistry 13:2633-2637 1974). Poly(A)+RNA was selectedfrom the total RNA using oligo d(T) cellulose chromatography (Aviv andLeder, Proc. Natl. Acad. Sci. USA 69:1408 (1972)).

First strand cDNA was synthesized from one time poly d(T)-selectedcerebellum poly(A)+RNA in two separate reactions. One reaction,containing radiolabeled dATP, was used to assess the quality of firststrand synthesis. The second reaction was carried out in the absence ofradiolabeled dATP and was used, in part, to assess the quality of secondstrand synthesis. Superscript reverse transcriptase (BRL) was usedspecifically as described below. A 2.5× reaction mix was prepared atroom temperature by mixing, in order, 10 μl of 5× reverse transcriptasebuffer (BRL; 250 mM Tris-HCl pH 8.3, 375 mM KC1, and 15 mM MgCl₂), 2.5μl 200 mM dithiothreitol (made fresh or stored in aliquots at -70° C.)and 2.5 μl of a deoxynucleotide triphosphate solution containing 10 mMeach of dATP, dGTP, dTTP and 5-methyl dCTP (Pharmacia). The reaction mixwas aliquoted into two tubes of 7.5 μl each. To the first tube, 1.3 μlof 10 μCi/μ l α³² P-dATP (Amersham) was added and 1.3 μl of water wasadded to the second reaction tube. Seven microliters from each tube wastransferred to reaction tubes. Fourteen microliters of a solutioncontaining 10 μg of cerebellum poly (A)+RNA diluted in 14 μl of 5 mMTris-HCl pH 7.4, 50 μM EDTA was mixed with 2 μl of 1 μg/μl first strandprimer, ZC2938 (Table 1; Sequence ID No. 11), and the primer wasannealed to the RNA by heating the mixture to 65° C. for 4 minutes,followed by chilling in ice water. Eight microliters of the RNA-primermixture was added to each of the two reaction tubes followed by 5 μl of200 U/μl Superscript reverse transcriptase (BRL). The reactions weremixed gently, and the tubes were incubated at 45° C. for 30 minutes.After incubation, 80 μl of 10 mM Tris-HC1 pH 7.4, 1 mM EDTA was added toeach tube, the samples were vortexed and centrifuged briefly. Threemicroliters of each reaction was removed to determine total counts andTCA precipitable counts (incorporated counts). Two microliters of eachsample was analyzed by alkaline gel electrophoresis to assess thequality of first strand synthesis. The remainder of each sample wasethanol precipitated. The nucleic acids were pelleted by centrifugation,washed with 80% ethanol and air dried for ten minutes. The first strandsynthesis yielded 1.4 μg of cerebellum cDNA or a 28% conversion of RNAinto DNA.

Second strand cDNA synthesis was performed on the RNA-DNA hybrid fromthe first strand reactions under conditions which encouraged firststrand priming of second strand synthesis resulting in DNA hairpinformation. The nucleic acid pellets containing the first strand cDNAwere resuspended in 71 μl of water. To assess the quality of secondstrand synthesis, α³² P-dATP was added to the unlabeled first strandcDNA. To encourage formation of the hairpin structure, all reagentsexcept the enzymes were brought to room temperature, and the reactionmixtures were set up at room temperature. (Alternatively, the reagentscan be on ice and the reaction mixture set up at room temperature andallowed to equilibrate at room temperature for a short time prior toincubation at 16° C.) Two reaction tubes were set up for each synthesis.One reaction tube contained the unlabeled first strand cDNA and theother reaction tube contained the radiolabeled first strand cDNA. Toeach reaction tube, 20 μl of 5× second strand buffer (100 mM Tris, pH7.4, 450 mM KC1, 23 mM MgCl₂, 50 mM (NH₄)₂ SO₄), 3 μl of beta-NAD and 1μl of a deoxynucleotide triphosphate solution containing 10 mM each ofdATP, dGTP, dTTP and dCTP (Pharmacia), 1 μl α³² P-dATP or 1 μl of water(the radiolabeled dATP was added to the tube containing the unlabeledfirst strand cDNA), 0.6 μl of 7 μl E. coli DNA ligase(Boehringer-Mannheim), 3.1 μl of 8 U/μl E. coli DNA polymerase I(Amersham), and 1 μl of 2 U/μl of RNase H (BRL). The reactions wereincubated at 16° C. for 2 hours. After incubation, 3 μl was taken fromeach reaction tube to determine total and TCA precipitable counts. Twomicroliters of each sample was analyzed by alkaline gel electrophoresisto assess the quality of second strand synthesis by the presence of aband of approximately twice unit length. To the remainder of eachsample, 2 μ l of 2.5 μg/μl oyster glycogen, 5 μl of 0.5 M EDTA and 200μl of 10 mM Tris-HC1 pH 7.4, 1 mM EDTA were added, the samples werephenol-chloroform extracted, and isopropanol precipitated. The nucleicacids were pelleted by centrifugation, washed with 80% ethanol and airdried. The yield of double stranded cDNA in each of the reactions wasapproximately 2 μg.

The single-stranded DNA in the hairpin structure was clipped using mungbean nuclease. Each second strand DNA sample was resuspended in 12 μl ofwater. Two microliters of 10× mung bean buffer (0.3 M NaOAC, pH 4.6, 3 MNaCl, 10 mM ZnSO₄), 2 μl of 10 mM dithiothreitol, 2 μl of 50% glycerol,and 2 μl of 10 U/μl mung bean nuclease (NEB, lot 7) were added to eachtube, and the reactions were incubated at 30° C. for 30 minutes. Afterincubation, 80 μl of 10 mM Tris-HC1 pH 7.4, 1 mM EDTA was added to eachtube, and 2 μl of each sample was subjected to alkaline gelelectrophoresis to assess the cleavage of the second strand product intounit length cDNA. One hundred microliters of 1 M Tris-HC1 pH 7.4 wasadded to each sample, and the samples were twice extracted withphenol-chloroform. Following the final phenol-chloroform extraction, theDNA was isopropanol precipitated. The DNA was pelleted bycentrifugation, washed with 80% ethanol and air dried. Approximately 2μg of DNA was obtained from each reaction.

The cDNA was blunt-ended with T4 DNA polymerase after the cDNA pelletswere resuspended in 12 μl of water. Two microliters of 10× T4 DNApolymerase buffer (330 mM Tris-acetate, pH 7.9, 670 mM KAc, 100 mM MgAc,1 mg/ml gelatin), 2 μl of 1 mM dNTP, 2 μl 50 mM dithiothreitol, and 2 μlof 1 U/μl T4 DNA polymerase (Boehringer-Mannheim) were added to eachtube. After an incubation at 15° C. for 1 hour, 180 μl of 10 mM Tris-HClpH 7.4, 1 mM EDTA was added to each sample, and the samples werephenol-chloroform extracted followed by isopropanol precipitation. ThecDNA was pelleted by centrifugation, washed with 80% ethanol and airdried. Eco RI adapters (Invitrogen, Cat. #N409-20) were ligated to theblunted cDNA after the DNA from each reaction was resuspended in 6.5 μlwater.

The first strand primer encoded an Sst I cloning site to allow the cDNAto be directionally cloned into an expression vector. The cDNA wasdigested with Sst I followed by phenol-chloroform extraction andisopropanol precipitation. After digestion, the cDNA was electrophoresedin a 0.8% low melt agarose gel, and the cDNA over 4.2 kb waselectroeluted using an Elutrap (Schleicher and Schuell, Keene, N.H.).The electroeluted cDNA in 500 μl of buffer was isopropanol precipitatedand the cDNA was pelleted by centrifugation. The cDNA pellet was washedwith 80% ethanol.

A cerebellum cDNA library was established by ligating the cDNA to theEco RI-Sst I digested, agarose gel purified pVEGT'.

Ten sublibraries of one million clones each were constructedrepresenting a library of ten million independent clones. To prepareeach sublibrary, 80 ng of linearized vector were ligated to 40 ng ofcDNA. After incubation at room temperature for 11 hours, 2.5 μg ofoyster glycogen and 80 μl of 10 mM Tris-HCl, 1 mM EDTA was added and thesample was phenol-chloroform extracted followed by ethanolprecipitation. The DNA was pelleted by centrifugation, and the DNApellet washed with 80% ethanol. After air drying, the DNA wasresuspended in 3 μl of water. Thirty-seven microliters ofelectroporation-competent DH10B cells (BRL) was added to the DNA andelectroporation was completed using a BioRad electroporation unit. Afterelectroporation, 4 ml of SOC (Maniatis et al.) was added to the cells,and 400 μl was spread on each of 10-150 mm LB ampicillin plates. Eachplate represented a sublibrary of 100,000 clones. After an overnightincubation, the cells were harvested by adding 10 ml of LB ampicillinmedia to each plate and scraping the cells into the media. Glycerolstocks and plasmid DNA were prepared from each plate. The librarybackground (vector without insert) was established at about 15%.

Detection of Glu_(G) R Activity from the cDNA Library

The Xenopus oocyte efficiently translates exogenously added mRNA.Preliminary experiments were done using the mouse ml muscarinic receptorcDNA (a G protein coupled receptor that can be detected byvoltage-clamp) cloned into pVEGT'. Injection of RNA transcribed in vitrofrom increasing dilutions of the ml template DNA indicated that mlagonist induced activity could be detected for one clone in a pool sizeof 100,000. A cerebellum sublibrary was plated into ten pools of 100,000unique clones.

The pools could also be replica plated onto a nitrocellulose filter andthe original and replica allowed to grow for a few hours. The originalplate is scraped to harvest all the colonies. Plasmid DNA is preparedand purified by cesium chloride gradient ultracentrifugation. The DNAfrom each pool is transcribed in vitro with T7 RNA polymerase in thepresence of 7-methyl-G, the capped nucleotide, to increase translationefficiency. Template DNA transcription reactions are spiked with adilution of two control genes cloned into pVEGT': the mouse ml gene anda secreted version of the human placental alkaline phosphatase gene(SEAP; Tate et al., Fed. Am. Soc. Exp. Biol. 8:227-231 (1990),incorporated by reference herein). Transcription from the control geneswould allow selection of oocytes that more efficiently translate theinjected RNA, and a determination whether oocytes that are negative forthe Glu_(G) R are true negatives, that is, still having a detectable mlagonist-induced response.

Plasmid DNA prepared from each of the 10 pools of 100,000 clones, whichin total represented one sublibrary of one million clones of thecerebellum cDNA library, was purified by cesium chloride gradientultracentrifugation. The DNA was transcribed in vitro with T7 RNApolymerase (Pharmacia) in the presence of capped nucleotide (GpppG,Pharamcia). The presence of a poly (A) sequence and two T7 RNApolymerase terminators in pVEGT' resulted in RNA with a capped 5' end,the sequence of the cDNA insert, and 3' poly (A) tails. Capped RNA isbelieved necessary for efficient translation in oocytes (Noma et al.Nature 319:640 (1986)) and the poly (A) sequence has been shown toincrease the synthesis of a protein in oocytes by more then 40 fold. Thetranscription reaction tubes were set up by adding 12 μl of 5×transcription buffer (Stratagene Cloning Systems, La Jolla, Calif.), 3μl each of 10 mM ATP, CTP, GTP, and UTP, 6 μl of 10 mM GpppG(Pharmacia), 6 μl of 1 mg/ml BSA, 3 μl of 200 mM DTT, 1.5 μl of 40 U/μlRNasin (ProMega Biotech, Madison, Wis.), 8.5 μl of water, 10 μl of cDNAcontaining 5 to 10 μg DNA, and 1 μl of 70 U/μl T7 RNA polymerase. Aftermixing, 10 μl of the reaction was transferred to a tube containing 0.5μCi of α³² P-UTP to determine the total counts and counts incorporatedinto RNA. The samples were incubated at 37° C. for one hour. The cDNA inthe unlabeled samples was degraded with the addition of 1 μl of 200 mMDTT, 2 μl of 30 U/μl DNase I, and 0.5 μl of 40 U/μl RNasin and theincubation was continued at 37° C. for 15 minutes. Forty microliters ofwater was added to the radiolabeled reactions, and 1 μl was removed fromeach sample and counted to determine total counts. The remainder of thelabeled samples were ethanol precipitated. The samples were centrifugedto collect the RNA and the RNA pellets were counted to determine thecounts incorporated into RNA. After the DNA degradation reaction in theunlabeled samples, 70 μl of 10 mM Tris-HCl, 1 mM EDTA was added to eachsample, and the samples were twice-extracted with phenol-chloroformfollowed by one chloroform extraction. The RNA was ethanol precipitated.After centrifugation to collect the RNA, the pellets were washed with80% ethanol, followed by air drying for 10 minutes. A typical yield ofthe unlabeled RNA was 20 to 30 μg. The unlabeled RNA was resuspended at2 μg/μl in diethylpyrocarbonate (DEPC, Sigma) treated water and storedat -70° C.

Prior to microinjection into oocytes, the RNA samples were thawed andcentrifuged in a microfuge for 5 minutes to remove any particles thatmight clog a microinjection pipet. After centrifugation, 80% of eachsample was removed and split into two tubes.

The RNA from each of the 10 sublibraries were injected into oocytes asdescribed above and translation was allowed for four days. Expression ofGlu_(G) R activity was assessed by voltage-clamp assay as describedabove. One of the 10 sublibraries, Z93-1.9, produced a signal withadministration of quisqualate to the oocyte.

Subdivision of the cDNA Library Pool to Obtain Pure Glu_(G) R Clone

The DNA pool (Z93-1.9) was subdivided by plating clones from theglycerol stock onto LB ampicillin plates. To determine the number ofclones that should be plated for the subdivision of the 100,000 clonepool to identify a positive clone, the probability equation N=ln (1-P)/ln (1-f) (Maniatis et al., ibid.) was used, where P is the desiredprobability of including the clone of interest, f is the fraction ofpositive clones in the pool, and N is the number of clones to be platedto provide the given probability. For a probability of 99.8% for a poolsize of 100,000 to contain one positive clone, 621,461 clones should beplated.

Forty-eight 150 mm LB ampicillin plates were plated with the glycerolstock representing the 100,000 positive pool, Z93-1.9, at a density ofapproximately 14,000 clones per plate to give a total of 670,000 clones.After an overnight incubation 37° C., the bacteria on each plate wereharvested into 10 ml of Solution I (as described by Birnboim and Doly,Nuc. ACids Res. 7:1513 (1979)), incorporated by reference herein). Aglycerol stock was prepared from a portion of the cells, and plasmid DNAwas prepared from the remainder of the cells. Six pools of DNArepresenting eight of the LB ampicillin plates each were prepared bycombining one tenth of the plasmid DNA from groups of eight plates intoeach pool. The plasmid DNA from these six pools was purified by cesiumchloride gradient centrifugation. The DNA was transcribed into RNA asoutlined above. Transcription of the parent pool Z95-1.9 was included asthe positive control. Oocytes were injected with the RNA andvoltage-clamp assays on the oocytes identified pool Z99-25 -32 aspositive for Glu_(G) R. Pool Z99-25-32 contained DNA prepared fromplates 25 through 32.

Plasmid DNA from plates 25 to 32 were cesium chloride banded andtranscribed into RNA as described above along with the positive parentpool Z99-25-32. Oocytes were injected with the RNA and voltage clampassays, carried out as described above, identified pools Z104-25 andZ111-32 as being weakly positive, Z106-27 and Z109-30 as intermediatelypositive, and Z108-29 and Z110-31 as the most positive. The poolresulting in Z110-31 was chosen for further subdivision.

Identification of positive pools from the subdivision of the positivepool of 14,000 (Z110-31) from the glycerol stock was unsuccessful.Therefore, plasmid DNA prepared from the pool resulting in Z110-31 waselectroporated into bacteria and plated on 60 plates at a density of1,000 clones/plate. Plasmid DNA was prepared from the bacteria harvestedfrom each plate. Aliquots of the plasmid DNA from each plate were mixedto make six pools representing ten plates each. The plasmid DNA wascesium chloride banded, and the RNA was transcribed as described above.RNA was transcribed from pools Z108-29, Z110-31, and a muscarinicreceptor cDNA, ml, for use as positive controls. The RNA was injectedinto oocytes and voltage-clamp assays were carried out as describedabove. The assays identified pool Z133-21 to 30 as positive.

Plasmid DNA from plates 21 to 30 were cesium chloride banded andtranscribed as described above. The transcribed RNA and the RNA from theparent pool Z133-21 to 30 were injected into oocytes and assayed asdescribed above. The voltage-clamp assay identified pool Z142-22 aspositive.

Identification of positive pools by the subdivision of the positive poolZ142-22 from a glycerol stock proved unsuccessful. Restriction analysisof plasmid DNA prepared from randomly selected clones from pools Z110-31(the pool of 14,000) and Z142-22 (the pool of 1,000) indicated that 50%of pool Z110-31 and 68% of pool Z142-22 were clones without inserts.

To assess physical methods for enriching for the Glu_(G) R clone and toestablish how many clones from pool Z142-22 needed to be assayed toinclude a Glu_(G) R clone, undigested plasmid DNA from pool Z142-22 waselectrophoresed on an agarose gel. The super-coil band representingvector without insert was cut out and the remainder of the DNA waseluted from the gel. The DNA was then electroporated into bacteriacells, and plated at densities of 3,400, 6,900, and 13,800 clones perplate. The plates were replica plated and grown overnight. Plasmid DNAwas prepared from the cells harvested from the replica of each plate.The plasmid DNA was transcribed, and the RNA was assayed in oocytes asdescribed above. As a control, each pool contained the equivalent of onecolony of ml as an internal positive control. In addition, ml was usedas an external positive control. The voltage-clamp assays identified theDNA from the 6,900 clone pool (Z167-7) as positive.

The clones represented on the 6,900 clone plate that resulted in thepositive pool Z167-7 were subdivided by replica plating the master plateonto a BIOTRANS nylon membrane (ICN flow, Costa Mesa Calif.) on an LBampicillin plate. The replica plate was incubated four hours at 37° C.After incubation, sub-pools were prepared by removing the membrane fromthe plate, taping the membrane to a sterile glass plate on a light box,and overlaying the membrane with a grid which divided the membrane into100 sections. The sections of the grid and underlying membrane were thencut out with a razor blade that had been dipped in alcohol and flamedbetween each cut. Alcohol-treated, flamed forceps were used to transfereach membrane section to a test tube containing 12.5 ml of LB ampicillinmedia. The cultures containing the membrane sections were incubatedovernight at 37° C. After incubation, 0.5 ml of each culture was mixedwith 0.5 ml of 50% glycerol and stored at -70° C. to establish glycerolstocks of each sub-pool. Aliquots of the 100 cultures were pooled in a10×10 matrix with samples (1) through (10) on the abscissa and samples(a) through (j) on the ordinate. For example, 1 ml of cultures (1)through (10) were added to tube 1 and 1 ml of cultures (1), (11), (21) ,(31) , (41) , (51) , (61), (71) , (81) , and (91) were added to tube (a)and so on until 10 rows of 10 and 10 columns containing pools of 10cultures each were completed. Ten microliters of an overnight culturecontaining ml-transformed bacteria was added to each pool as an internalcontrol. Plasmid DNA was prepared from the 20 sub-pools, and the DNA waspurified by cesium chloride gradient centrifugation. RNA was transcribedfrom the plasmid DNA and was assayed in oocytes as described above.Positive controls were the parent pool Z167-7 and pure ml RNA. Thevoltage-clamp assays indicated that only pools Z175-1 and Z191-g werepositive. Consulting the matrix,this indicated that the membrane sectionnumber (7) contained the Glu_(G) R clone.

To subdivide the clones contained in section (7), a piece of Biodyne Amembrane was applied to the master plate containing section (7), themembrane extending beyond section (7) on each side by half the width ofsection (7). The membrane was removed from the plate, applied to a freshLB ampicillin plate colony side up, and incubated overnight at 37° C.The membrane was subdivided as described above with the central regionof the membrane, the actual section (7) area, divided into 9 small,equivalent-sized squares and the membrane on each side of section (7)was taken as four additional areas. Each membrane section was used toinoculate a 10 ml liquid culture. Bacteria transformed with the ml clonewere used as an internal control in each culture as described above.After overnight incubation at 37° C., plasmid DNA was prepared, and theDNA was purified by cesium chloride gradient centrifugation. RNA wastranscribed and assayed in oocytes as described above using RNA from mland the parent pool number (7) as positive controls. Glu_(G) R activitywas found in only pool Z203-7 corresponding to membrane section number(7).

Pool Z203-7 was subdivided by electroporating the plasmid DNA preparedfrom the membrane section number (7) into DH10Belectroporation-competent cells. The transformants were plated at adensity enabling individual colonies to be picked. Individuals cloneswere picked to a master plate and into 2 ml of LB ampicillin media. Thecultures were incubated overnight, and plasmid DNA was prepared by themethod essentially described by Holms and Quigley (Anal. Bioc. 114: 193,(1981)). Restriction analysis suggested that the clones were groupedinto 7 different classes of clones. Plasmid DNA, prepared from eachclass, representing fifty total clones were prepared, transcribed, andassayed in oocytes as described above. However, none of the clones werepositive.

To screen for positive clones, electroporation-competent E. coli DH10Bcells were electroporated with the DNA prepared from membrane sectionnumber (7) (Z203-7) and were plated at 180, 360, 900, and 1800 coloniesper plate. The plates were incubated overnight, and replica plates wereprepared as described above. Plasmid DNA prepared from each replicaplate was combined with 1 to 1000 parts of ml as an internal control.The DNA pools, the ml clone and the parent pool Z203-7 were transcribed,and the RNA was assayed by oocyte injection. The first transcription andinjection showed no positives, however, upon retranscription andreanalysis the 1800 clone pool (Z264-1800) was positive for Glu_(G) Ractivity.

To subdivide the positive pool of 1800 (Z264-1800), all of the coloniesfrom the plate of 1800, 1528 in total, were each picked to 16 100 mm LBampicillin agar plates on a 100 colony grid. After overnight growth, oneset of the duplicate plates was designated as a master set and wasplaced at 4° C. The other set was replica plated to a third set ofplates. After overnight incubation of these plates, the cells on thereplica plates were harvested into media and plasmid DNA was preparedfrom the pooled cells. As described above, an internal ml control wasincluded in each DNA preparation. m1 DNA and the parent Z264-1800 DNAwere used as external positive controls. Plasmid DNA prepared from the16 plates was transcribed, and the RNA was assayed in oocytes asdescribed above. One of the pools of 100 clones, Z256-I produced Glu_(G)R activity.

To identify which clone of the 100 clones from Z256-I produced theGlu_(G) R activity, a 10×10 matrix of the clones was constructed. Aliquid culture of each clone was grown. One milliliter of each culturewas added to each of two tubes representing the appropriate row andcolumn of the 10×10 matrix. As described previously, plasmid DNAencoding ml was used as an internal positive control. Plasmid DNAprepared from each tube, ml DNA and DNA from the parent pool Z264-1800were transcribed and assayed in oocytes as described above. Glu_(G) Ractivity was identified only in row (5) and column (e). Thus, thepositive clone number 45 was identified as containing the Glu_(G) Ractivity.

To confirm the result, plasmid DNA from clone #45 was prepared,transcribed and assayed in oocytes as described above. The results ofthe assay indicated that clone #45 was capable of producing Glu_(G) Ractivity. FIG. 2 illustrates the data taken from voltage-clamprecordings at several stages in the subfractionation of the cerebellumlibrary. Panel (a) is a recorded response to quisqualate of an oocytepreviously injected with in vitro transcribed RNA from a rat cerebellumsublibrary of 100,000 independent colonies; panel (b) shows the responseto quisqualate in a cell previously injected with RNA transcribed from asubfractionated pool of 14,000 colonies. The peak current was truncatedby the chart recorder, but the actual peak current (estimated from adigital panel meter) was approximately 1300 nA. Panel (c) shows theresponse to quisqualate in a cell injected with pure Glu_(G) R RNA fromclone 45-A. The amount of RNA injected per oocyte was approximately 100ng, except in panel (c) where the amount of RNA was 50 pg.

The following describes an alternative means for subdividing andscreening a positive pool. Working with cDNA inserts in a plasmid basedrather than a lambda-based vector influences the subfractionationprotocol. Once a positive pool is identified, the replica filter isoverlayed with another sterile nitrocellulose filter. The filter is cutinto 88 pieces by using evenly spaced cuts of 10 rows and 10 columns toform a grid. Each of the 88 pieces is transferred to 10 ml of sterileLB+Amp and grown for several hours. Twenty pools are formed; C 1-10(corresponding to column number) and R 1-10 (corresponding to rownumber). An aliquot of each of the 88 subfractions is pipetted into 2tubes, corresponding to its position in a row and a column. DNA isisolated from the 20 pools, purified on CsCl gradients and transcribedin an in vitro reaction that includes the control ml and SEAP plasmids.After injection into oocytes and voltage-clamp recording there are 2positive pools, pinpointing the location of 1 of the 88 originalsubfractions.

Because the positive clone is still part of a pool it must be furthersubdivided. The probability equation described above is used todetermine the number of clones to be plated for the next subdivision ofthe pool. The glycerol stock from the positive pool is plated out at,e.g., 3000, 6000 and 18,000 clones per plate. After replica plating theDNA is harvested, transcribed, injected and assayed. The pool which ispositive is subdivided into a grid of 88 as described above. The assayis repeated, and a single square of the grid is positive. At the nextstep of subdivision of the pool, 100 individual colonies to a plate arepicked, replica plated, and 20 pools are made for transcription andassay. Positive clones are streaked out, several colonies picked andrestriction mapped and template and transcript prepared for injectionand assay.

Characterization of Glu_(G) R

To establish that the Glu_(G) R encoded by clone 45-A couples toG-protein, clone 45-A Glu_(G) R RNA was transcribed and injected intooocytes as described above. Two days after injection the oocytes weredivided into control and toxin-treated groups. The oocytes in thetoxin-treated group were treated with a final concentration of 4 μg/mlof B. pertussis toxin (List Biological Laboratories Inc., Campbell,Calif.), and both groups were incubated for 24 hours at 19° C. asdescribed by Sugiyama et al., Nature 325:531 (1987) and Moriarty et al.,J. Biol. Chem. 264:13521 (1989), both of which are incorporated byreference herein. The oocytes from both the control and toxin-treatedgroups were subjected to voltage-clamp assays as described previously.In one example, oocytes perfused as described previously with 100 μML-glutamic acid showed a mean L-glutamic acid-induced current of 264.2nA±73 nA in control oocytes (SEM, n=6) and 57.7 nA±19 nA (n=9) intoxin-treated oocytes. The mean membrane current in the toxin-treatedgroup was significantly smaller (p<0.01) than in the control groupsuggesting that oocytes injected with 45-A RNA coupled to a pertussistoxin-sensitive G protein.

L-glutamic acid and some of its structural derivatives that are known toactivate Glu_(G) R currents in a dose-dependent manner were applied tooocytes that had been injected with RNA transcribed from the 45-A clone.RNA was transcribed and oocytes were prepared and injected as previouslydescribed. Dose dependent responses were measured using voltage clampassays were carried out in the presence of increasing concentrations ofL-glutamic acid (Sigma), quisqualic acid (Sigma), ibotenic acid (Sigma),or trans 1-amino-cyclopentyl-1,3 dicarboxylic acid (tACPD; TocrisNeuramin, Essex, England). Four or five separate oocytes were perfusedwith increasing concentrations of a particular drug with 30 minutesbetween consecutive applications of the drug to minimize anyinterference from desensitization. The responses were normalized to asubsequent response to 100 μM L-glutamic acid. The data were analyzedusing the following equation:

    (Fractional current)=(Dose.sup.n)/(Dose.sup.n)+(EC.sub.50).sup.n,

where:

Dose=a dose of drug normalized to that evoked by a subsequentapplication of 100 μM L-glutamic acid;

Fractional current=the peak current evoked by a dose, as defined above;

EC₅₀ =effective concentration that evokes a 50% response (a measure ofthe potency of an agonist); and

n=the Hill coefficient, a measure of the cooperativity of the reaction.

Using this equation, the effective concentration at 50% stimulationrelative to 100 μM L-glutamic acid was determined for each dose responseexperiment. FIG. 6 shows a representative dose response curve forvarying concentrations of L-glutamic acid. The potency series ofglutamate analogs and their associated EC₅₀ 's are listed in Table 2.

                  TABLE 2                                                         ______________________________________                                        Glutamate Analog Potencies (EC.sub.50)                                        ______________________________________                                        Quisqualic acid      0.681  μM                                             L-glutamic acid      12.32  μM                                             Ibotenic acid        32.37  μM                                             tACPD                376    μM                                             ______________________________________                                    

In addition, oocytes were exposed to the following L-glutamic acidanalogs: aspartic acid (Tocris Neuramin), kainic acid,N-methyl-D-aspartic acid (NMDA; Sigma), 2-amino-4-phosphonobytyric acid(APB; Sigma), α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid(AMPA; Research Biochemicals Inc., Wayland, Mass.) at saturatingconcentrations and the responses were each normalized to a subsequentresponse to 100 μM L-glutamate. The L-glutamic acid analogs that werefound to be ineffective were 1 mM aspartic acid, 1 mM kainic acid, 100μM NMDA+10 μM glycine, 100 μM APB and 100 μM AMPA.

Voltage clamp assays were also carried out on injected oocytes tomeasure the inhibition by the putative glutamate G protein coupledreceptor antagonist, 2-amino-3-phosphonopropionic acid (AP3). Voltageclamp assays showed that at 1 mM, DL-AP3 (Sigma) reduced the currentevoked by 10 μM glutamic acid to 59.3±7.3% of the control.

Clone 45 cells were streaked out on LB Amp plates and several colonieswere picked, grown up and the DNA isolated. Pure 45-A DNA was preparedand restriction mapped by standard procedures. Clone 45-A has beendeposited with the American Type Culture Collection, 12301 ParklawnDrive, Rockville, Md., 20852, under ATCC Accession No. 68497. DNA wasdigested with single or multiple enzymes. The fragments were separatedon both 1% agarose and 4% Nusieve gels by electrophoresis. Afterelectrophoresis the DNA was transferred to nitrocellulose filters usingstandard protocols for Southern transfer. Restriction sites were mappedbased on size and based on hybridization to Pst I subclones of 45-A DNA.Additionally, the entire 45-A cDNA insert can be isolated by digestionwith Not I restriction endonuclease. The Not I insert was kinased with³² -P ATP, and after digestion of half of the sample with Bam HI toremove the 3' label, both samples were subjected to digestion with anumber of enzymes known to be present once in the insert. In this waythe unique sites could be localized. A restriction map of Glu_(G) Rclone 45-A is shown in FIG. 3.

The entire 45-A clone was sequenced in both directions using thedideoxynucleotide chain termination method (Sanger and Coulson, J Mol.Biol. 94:441 (1975), incorporated herein by reference). FIG. 5 (SequenceID Nos. 1 and 2) shows the DNA sequence and deduced amino acid sequenceof clone 45-A. FIG. 5 also shows the location of putative N-linkedglycosylation sites, which have been predicted to occur at the aminoacid sequence Asn-X-Thr.

As shown in FIG. 5, seven putative transmembrane domains have beenpredicted from the deduced amino acid sequence of clone 45-A using themethod described by Eisenberg et al. J. Mol. Biol. 179:125-142, (1984),incorporated herein by reference. Only those predicted to betransmembrane multimeric domains were included. An additionaltransmembrane domain (the third) was predicted using the method of Hoppand Woods, Proc. Natl. Acad. Sci. USA 78:3824-3838 (1981). Based onthese predictions, the protein encoded by clone 45-A appears to have twounusually large domains on the amino- and carboxy-termini that are notfound in any of the other reported G protein coupled receptors whichhave the common structural feature of seven predicted membrane spanningregions. Analysis of the deduced amino acid sequence of clone 45-Apredicts three other hydrophobic stretches including one at theamino-terminus of the sequence. This amino-terminal hydrophobic stretchmay be a signal sequence, although no signal cleavage site is predicteddownstream of the sequence.

Poly (A)+RNA was isolated from total rat brain and rat cerebellum usingoligo d(T) cellulose chromatography as described by Aviv and Leder(ibid.). Poly (A)+RNA from rat retina, rat heart, rat lung, rat liver,rat kidney, rat spleen, rat testis, rat ovary and rat pancreas werepurchased from Clonetech. The poly(A)+RNA samples were analyzed bynorthern analysis (Thomas, Proc. Natl., Acad. Sci. USA 77:5201-5205(1980), which is incorporated by reference herein). The RNA wasdenatured in glyoxal, electrophoresed in agarose and transferred to anitrocellulose membrane essentially as described by Thomas (ibid.). Thenorthern blot was hybridized with a radiolabeled 3473 bp Eco RI-Xba Ifragment from the 45-A clone. Autoradiography of the blot showedhybridization to a major band of approximately 7 kb and a smaller bandof approximately 3.8 kb in the total rat brain and rat cerebellum RNA.

Single-stranded cDNA was synthesized using 1 μg of the poly (A)+RNAusing Superscript reverse transcriptase (BRL) under conditions describedby the manufacturer. One fourth of the cDNA was used as a template forPCR amplification using 40 pmoles each of the Glu_(G) R-specific primersZC3652 (Table 1; Sequence ID Number 14) and ZC3654 (Table 1; Sequence IDNumber 15) and 2.5 U Taq I polymerase (Perkin Elmer Cetus, Norwalk. Va.)and conditions specified by the manufacturer. As an internal control,the PCR reaction also contained 2 pmoles each of the glucose-6-phosphatedehydrogenase-specific primers ZC3015 (Table 1; Sequence ID Number 12)and ZC3016 (Table 1; Sequence ID Number 13). After thirty cycles (oneminute at 94° C., one minute at 60° C., ninety seconds at 72° C.), thesamples were phenol-chloroform extracted and 20% of each reaction waselectrophoresed in agarose. The DNA was bidirectionally transferred tonitrocellulose membranes, and the filters were hybridized with eitherradiolabeled ZC3652, ZC3654, ZC3015 and ZC3016 (Sequence ID Nos. 14, 15,12 and 13, respectively) or with the radiolabeled Eco RI-Xba I fragmentof clone 45-A described above. Autoradiography of the hybridized blotshowed that Glu_(G) R transcript was mainly confined to total rat brainand rat cerebellum; however, longer exposures showed a Glu_(G)R-specific transcript in both retina and testis.

Total RNA was prepared, as described above, from specific rat brainregions including frontal cortex, cerebellum, hippocampus, cortex,striatum, pons medulla, and the remainder of the brain. Single-strandedcDNA was synthesized as described previously using 20 μg of total RNA in50 μl using Superscript reverse transcriptase (BRL) under conditionsdescribed by the manufacturer. After a one hour incubation at 42° C.,the samples were treated with RNAse (Boehringer Mannheim Biochemicals,Indianapolis, Ind.), phenol-chloroform extracted, and ethanolprecipitated. The samples were resuspended in water and half of eachsample was subjected to PCR amplification. Each PCR amplificationcontained 40 pmoles of each of the Glu_(G) R-specific primers ZC3652 andZC3654 described above (Sequence ID Numbers 14 and 15), 2 pmoles of eachof the glucose-6-phosphate dehydrogenase-specific primers ZC3015 andZC3016 (Sequence ID Nos. 12 and 13) and 2.5 U Taq I polymerase (PerkinElmer Cetus) and conditions described by the manufacturer. After 35cycles (one minute at 94° C., one minute at 60° C., ninety seconds at72° C.), the samples were phenol-chloroform extracted, and 20% of eachreaction was electrophoresed in agarose. The DNA was transferred to anitrocellulose membrane, and the filter was hybridized with theradiolabeled Eco RI-Xba I fragment of clone 45-A described above.Autoradiography of the hybridized blots showed a broad distribution ofthe Glu_(G) R transcript throughout the brain, although the frontalcortex and cerebellum appear to be somewhat enriched.

Southern analysis of rat and human genomic DNA was carried out using themethod essentially described by Blin et al. (Nuc. Acids Res. 3:2303(1976), which is incorporated by reference herein). Briefly, rat andhuman genomic DNA was prepared from the rat cell line UMR 106 (ATCC CRL1661) and a human hepatoma cell line (ATCC HTB 52), respectively. Thegenomic DNA was digested with either Eco RI or Pst I, andelectrophoresed through agarose. The DNA was transferred to anitrocellulose membrane, and the membrane was hybridized with aradiolabeled 1.6 kb Pst I fragment from clone 45-A. Autoradiography ofthe hybridized blot suggest that the human gene has a similar sequenceto the rat Glu_(G) R sequence, the Glu_(G) R gene contains at least oneintron, and that there are a small number of closely related genes.

Expression in Mammalian Cells

The entire Glu_(G) R cDNA insert was removed from the pVEGT' cloningvector by digestion with Not I and Xba I. The ends were blunted with DNApolymerase I (Klenow fragment) and dNTPs, and were then ligated with EcoRI (Smart) linkers. After linker ligation, the insert with Eco RI endsin kinased and ligated to Eco RI-cut and capped Zem228 expressionvector. Bacteria are transformed with the ligation reaction and clonesare characterized by restriction analysis and partial sequencing (seeFIG. 4).

Cultured mammalian cells, such as BHK 570 and BHK ts13 serve as hostcells for expression. Twenty-five μg of CsCl-purified DNA isprecipitated with calcium phosphate and added to tissue culture cells ina 150 mm plate. After 4 hours the cells are subjected to a glycerolshock and then put into non-selective medium. In some cases it may benecessary to include an antagonist to the Glu_(G) R in the medium. Thisis to prevent expression of a cytotoxic response in those cells wherethe Glu_(G) R is expressed at levels high enough to cause a certainamount of autoactivation. For assay of transiently expressed Glu_(G) Rligand binding activity or PLC activation, cells are harvested after 48hours. Stable expression is detected after 2 weeks of selection. TheZem228 expression vector includes a promoter capable of directing thetranscription of the Glu_(G) R gene, and a selectable marker for thebacterial neomycin resistance gene. Resistance to the drug G-418, aninhibitor of protein synthesis, is used to identify stably transfectedclones. Presence of the SV 40 ori region on the vector allows theexpression construction to also be used for transient expression. Insome instances it is preferable to include DNA for another selectablemarker, the DHFR gene, in the transfection protocol. Selection with bothG-418 and methotrexate allows isolation of clones whose expression ofGlu_(G) R can be subsequently amplified by the addition of increasinglyhigher concentrations of methotrexate to the culture medium.

Transfected cell lines expressing Glu_(G) R are identified by thebinding of 3H-glutamate to cells grown in tissue culture. Cell linesexpressing low to moderate levels of Glu_(G) R are used to set upfunctional screening assays.

BHK 570 cells expressing the rat G protein coupled glutamate receptorcDNA are plated into 24-well culture dishes at about 100,000 cells perwell. After 24 hours, the cells are rinsed with cold PBS and thenincubated in serum-free medium containing 3H-glutamate with or without a10-fold excess of unlabeled quisqualate. After 30 minutes at 37° C. thecells are chilled on ice and then harvested with trypsin directly ontoglass filters using an LKB 1295-001 automated cell harvester. Thesamples are rapidly washed with cold PBS, the filters dried and counted.

Functional Screening of Agonists and Antagonists

BHK 570 cells expressing Glu_(G) R or mock transfected BHK 570 cells areplated into 24-well tissue culture dishes at about 100,000 cells perwell. After 24 hours, the cells are labeled with 0.2 μCi myo-(2-3H)inositol (specific activity=20 Ci/mmol; New England Nuclear) per well.At the end of the 24 hour incubation, the cells are washed withprewarmed Krebs-Henselsit buffer (equilibrated with 95% O₂ / 5% CO₂ atpH 7.4) containing 10 mM LiCl, and incubated for 5 minutes at 37° C. Theselected drugs are then added and the cells incubated for an additional30 minutes at 37° C. The reaction is stopped and the cells are lysed byaspirating off the media and adding 1 ml of fresh buffer and 1 ml ofice-cold perchloric acid. The cell lysate is then centrifuged anddiluted with an EDTA solution. The samples are then neutralized with amixture of freon/tri-n-octylamine, and the resulting supernatant dilutedwith water and applied to an Amprep minicolumn (Amersham RPN1908).Inositol phosphates are then eluted off the column and samples arecounted in a scintillation counter. A positive response is indicated byan increase in labeled inositol phosphate levels.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 15                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4300 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Rattus norvegicus                                               (F) TISSUE TYPE: Cerebellum                                                   (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: 45-A                                                               (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 377..3973                                                       (D) OTHER INFORMATION:                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       CCGAGAACGGCTGCAGTCCTCTGACCTGAGACCAATAGCTGTGTCTACCCGGACTCAGCG 60               TCCAGCTCACCGCCACTAACGCGCCGCGCATTGGACACCTGATCCACACACCTTCGGGCA120               CCAGTGAAAAACCGCGACTTGATTTTCTGGAAGAACGCCCCCAGGGTGTGGGAGCGGTCG180               TGGAGGACCAGCAGGAGGAAGCGGAGGGGAGAGGGGCAGTAGTG GAGGCAGAGAAAGCGT240              TGAACCAGCTGTGTTGGCCGAAGGCACGAAACGGCAAAAGGCAGCGGTGAGCATCTGTGT300               GGTTCCCGCTGGGAACCTGCAGGCAGGACCGGCGTGGGAACGTGGCTGGCCCGCGGTGGA360               CCGCGTCTTCGCCACAATGGTCCGG CTCCTCTTGATTTTCTTCCCAATG409                         MetValArgLeuLeuLeuIlePhePheProMet                                             1510                                                                          ATCTTTTTGGAGATGTCCATTTTGC CCAGGATGCCTGACAGAAAAGTA457                          IlePheLeuGluMetSerIleLeuProArgMetProAspArgLysVal                              152025                                                                        TTGCTGGCAGGTGCCTCGTCCCAGCGCTC CGTGGCGAGAATGGACGGA505                          LeuLeuAlaGlyAlaSerSerGlnArgSerValAlaArgMetAspGly                              303540                                                                        GATGTCATCATCGGAGCCCTCTTCTCAGTCCATCAC CAGCCTCCAGCC553                          AspValIleIleGlyAlaLeuPheSerValHisHisGlnProProAla                              455055                                                                        GAGAAGGTACCCGAAAGGAAGTGTGGGGAGATCAGGGAACAGTAT GGT601                          GluLysValProGluArgLysCysGlyGluIleArgGluGlnTyrGly                              60657075                                                                      ATCCAGAGGGTGGAGGCCATGTTCCACACGTTGGATAAGATTA ACGCG649                          IleGlnArgValGluAlaMetPheHisThrLeuAspLysIleAsnAla                              808590                                                                        GACCCGGTGCTCCTGCCCAACATCACTCTGGGCAGTGAGATCCG GGAC697                          AspProValLeuLeuProAsnIleThrLeuGlySerGluIleArgAsp                              95100105                                                                      TCCTGCTGGCACTCTTCAGTGGCTCTCGAACAGAGCATCGAATTCATC 745                          SerCysTrpHisSerSerValAlaLeuGluGlnSerIleGluPheIle                              110115120                                                                     AGAGACTCCCTGATTTCCATCCGAGATGAGAAGGATGGGCTGAACCGA793                            ArgAspSerLeuIleSerIleArgAspGluLysAspGlyLeuAsnArg                             125130135                                                                     TGCCTGCCTGATGGCCAGACCCTGCCCCCTGGCAGGACTAAGAAGCCT841                           CysLeuProA spGlyGlnThrLeuProProGlyArgThrLysLysPro                             140145150155                                                                  ATTGCTGGAGTGATCGGCCCTGGCTCCAGCTCTGTGGCCATTCAAGTC889                           IleAlaGl yValIleGlyProGlySerSerSerValAlaIleGlnVal                             160165170                                                                     CAGAATCTTCTCCAGCTGTTCGACATCCCACAGATCGCCTATTCTGCC937                           GlnAsnLeu LeuGlnLeuPheAspIleProGlnIleAlaTyrSerAla                             175180185                                                                     ACAAGCATAGACCTGAGTGACAAAACTTTGTACAAATACTTCCTGAGG985                           ThrSerIleAsp LeuSerAspLysThrLeuTyrLysTyrPheLeuArg                             190195200                                                                     GTGGTCCCTTCTGACACTTTGCAGGCAAGGGCGATGCTCGACATAGTC1033                          ValValProSerAspThrL euGlnAlaArgAlaMetLeuAspIleVal                             205210215                                                                     AAGCGTTACAACTGGACCTATGTCTCAGCAGTCCACACAGAAGGGAAT1081                          LysArgTyrAsnTrpThrTyrValSerAl aValHisThrGluGlyAsn                             220225230235                                                                  TACGGCGAGAGTGGAATGGATGCTTTCAAAGAACTGGCTGCCCAGGAA1129                          TyrGlyGluSerGlyMetAspAlaPhe LysGluLeuAlaAlaGlnGlu                             240245250                                                                     GGCCTCTGCATCGCACACTCGGACAAAATCTACAGCAATGCTGGCGAG1177                          GlyLeuCysIleAlaHisSerAspLys IleTyrSerAsnAlaGlyGlu                             255260265                                                                     AAGAGCTTTGACCGGCTCCTGCGTAAACTCCGGGAGCGGCTTCCCAAG1225                          LysSerPheAspArgLeuLeuArgLysLeuA rgGluArgLeuProLys                             270275280                                                                     GCCAGGGTTGTGGTCTGCTTCTGCGAGGGCATGACAGTGCGGGGCTTA1273                          AlaArgValValValCysPheCysGluGlyMetThrVa lArgGlyLeu                             285290295                                                                     CTGAGTGCCATGCGCCGCCTGGGCGTCGTGGGCGAGTTCTCACTCATT1321                          LeuSerAlaMetArgArgLeuGlyValValGlyGluPheSerLeuIle                              300305310315                                                                  GGAAGTGATGGATGGGCAGACAGAGATGAAGTCATCGAAGGCTATGAG1369                          GlySerAspGlyTrpAlaAspArgAspGluValIleGluGlyTyr Glu                             320325330                                                                     GTGGAAGCCAACGGAGGGATCACAATAAAGCTTCAGTCTCCAGAGGTC1417                          ValGluAlaAsnGlyGlyIleThrIleLysLeuGlnSerProGluV al                             335340345                                                                     AGGTCATTTGATGACTACTTCCTGAAGCTGAGGCTGGACACCAACACA1465                          ArgSerPheAspAspTyrPheLeuLysLeuArgLeuAspThrAsnThr                               350355360                                                                    AGGAATCCTTGGTTCCCTGAGTTCTGGCAACATCGCTTCCAGTGTCGC1513                          ArgAsnProTrpPheProGluPheTrpGlnHisArgPheGlnCysArg                              365 370375                                                                    CTACCTGGACACCTCTTGGAAAACCCCAACTTTAAGAAAGTGTGCACA1561                          LeuProGlyHisLeuLeuGluAsnProAsnPheLysLysValCysThr                              380385 390395                                                                 GGAAATGAAAGCTTGGAAGAAAACTATGTCCAGGACAGCAAAATGGGA1609                          GlyAsnGluSerLeuGluGluAsnTyrValGlnAspSerLysMetGly                              400 405410                                                                    TTTGTCATCAATGCCATCTATGCCATGGCACATGGGCTGCAGAACATG1657                          PheValIleAsnAlaIleTyrAlaMetAlaHisGlyLeuGlnAsnMet                              415 420425                                                                    CACCATGCTCTGTGTCCCGGCCATGTGGGCCTGTGTGATGCTATGAAA1705                          HisHisAlaLeuCysProGlyHisValGlyLeuCysAspAlaMetLys                              430 435440                                                                    CCCATTGATGGCAGGAAGCTCCTGGATTTCCTCATCAAATCCTCTTTT1753                          ProIleAspGlyArgLysLeuLeuAspPheLeuIleLysSerSerPhe                              445450 455                                                                    GTCGGAGTGTCTGGAGAGGAGGTGTGGTTCGATGAGAAGGGGGATGCT1801                          ValGlyValSerGlyGluGluValTrpPheAspGluLysGlyAspAla                              460465470 475                                                                 CCCGGAAGGTATGACATTATGAATCTGCAGTACACAGAAGCTAATCGC1849                          ProGlyArgTyrAspIleMetAsnLeuGlnTyrThrGluAlaAsnArg                              480485 490                                                                    TATGACTATGTCCACGTGGGGACCTGGCATGAAGGAGTGCTGAATATT1897                          TyrAspTyrValHisValGlyThrTrpHisGluGlyValLeuAsnIle                              495500 505                                                                    GATGATTACAAAATCCAGATGAACAAAAGCGGAATGGTACGATCTGTG1945                          AspAspTyrLysIleGlnMetAsnLysSerGlyMetValArgSerVal                              510515520                                                                     TGCAGTGAGCCTTGCTTAAAGGGTCAGATTAAGGTCATACGGAAAGGA1993                          CysSerGluProCysLeuLysGlyGlnIleLysValIleArgLysGly                              525530535                                                                     GAAGTGAG CTGCTGCTGGATCTGCACGGCCTGCAAAGAGAATGAGTTT2041                         GluValSerCysCysTrpIleCysThrAlaCysLysGluAsnGluPhe                              540545550555                                                                  GTGCAG GACGAGTTCACCTGCAGAGCCTGTGACCTGGGGTGGTGGCCC2089                         ValGlnAspGluPheThrCysArgAlaCysAspLeuGlyTrpTrpPro                              560565570                                                                     AACGCA GAGCTCACAGGCTGTGAGCCCATTCCTGTCCGTTATCTTGAG2137                         AsnAlaGluLeuThrGlyCysGluProIleProValArgTyrLeuGlu                              575580585                                                                     TGGAGTGACA TAGAATCTATCATAGCCATCGCCTTTTCTTGCCTGGGC2185                         TrpSerAspIleGluSerIleIleAlaIleAlaPheSerCysLeuGly                              590595600                                                                     ATCCTCGTGACGCTGTT TGTCACCCTCATCTTCGTTCTGTACCGGGAC2233                         IleLeuValThrLeuPheValThrLeuIlePheValLeuTyrArgAsp                              605610615                                                                     ACACCCGTGGTCAAATCCTCCAGTAGG GAGCTCTGCTATATCATTCTG2281                         ThrProValValLysSerSerSerArgGluLeuCysTyrIleIleLeu                              620625630635                                                                  GCTGGTATTTTCCTCGGCTATGTG TGCCCTTTCACCCTCATCGCCAAA2329                         AlaGlyIlePheLeuGlyTyrValCysProPheThrLeuIleAlaLys                              640645650                                                                     CCTACTACCACATCCTGCTACCTCC AGCGCCTCCTAGTTGGCCTCTCT2377                         ProThrThrThrSerCysTyrLeuGlnArgLeuLeuValGlyLeuSer                              655660665                                                                     TCTGCCATGTGCTACTCTGCTTTAGTGAC CAAAACCAATCGTATTGCA2425                         SerAlaMetCysTyrSerAlaLeuValThrLysThrAsnArgIleAla                              670675680                                                                     CGCATCCTGGCTGGCAGCAAGAAGAAGATCTGCACC CGGAAGCCCAGA2473                         ArgIleLeuAlaGlySerLysLysLysIleCysThrArgLysProArg                              685690695                                                                     TTCATGAGCGCTTGGGCCCAAGTGATCATAGCCTCCATTCTGATT AGT2521                         PheMetSerAlaTrpAlaGlnValIleIleAlaSerIleLeuIleSer                              700705710715                                                                  GTACAGCTAACACTAGTGGTGACCTTGATCATCATGGAGCCTC CCATG2569                         ValGlnLeuThrLeuValValThrLeuIleIleMetGluProProMet                              720725730                                                                     CCCATTTTGTCCTACCCGAGTATCAAGGAAGTCTACCTTATCTG CAAT2617                         ProIleLeuSerTyrProSerIleLysGluValTyrLeuIleCysAsn                              735740745                                                                     ACCAGCAACCTGGGTGTAGTGGCCCCTGTGGGTTACAATGGACTCCTC 2665                         ThrSerAsnLeuGlyValValAlaProValGlyTyrAsnGlyLeuLeu                              750755760                                                                     ATCATGAGCTGTACCTACTATGCCTTCAAGACCCGCAACGTGCCGGCC2713                           IleMetSerCysThrTyrTyrAlaPheLysThrArgAsnValProAla                             765770775                                                                     AACTTCAATGAGGCTAAATACATCGCCTTCACCATGTACACTACCTGC2761                          AsnPheAsnG luAlaLysTyrIleAlaPheThrMetTyrThrThrCys                             780785790795                                                                  ATCATCTGGCTGGCTTTCGTTCCCATTTACTTTGGGAGCAACTACAAG2809                          IleIleTr pLeuAlaPheValProIleTyrPheGlySerAsnTyrLys                             800805810                                                                     ATCATCACTACCTGCTTCGCGGTGAGCCTCAGTGTGACGGTGGCCCTG2857                          IleIleThr ThrCysPheAlaValSerLeuSerValThrValAlaLeu                             815820825                                                                     GGGTGCATGTTTACTCCGAAGATGTACATCATCATTGCCAAACCTGAG2905                          GlyCysMetPhe ThrProLysMetTyrIleIleIleAlaLysProGlu                             830835840                                                                     AGGAACGTCCGCAGTGCCTTCACGACCTCTGATGTTGTCCGCATGCAC2953                          ArgAsnValArgSerAlaP heThrThrSerAspValValArgMetHis                             845850855                                                                     GTCGGTGATGGCAAACTGCCGTGCCGCTCCAACACCTTCCTCAACATT3001                          ValGlyAspGlyLysLeuProCysArgSe rAsnThrPheLeuAsnIle                             860865870875                                                                  TTCCGGAGAAAGAAGCCCGGGGCAGGGAATGCCAATTCTAACGGCAAG3049                          PheArgArgLysLysProGlyAlaGly AsnAlaAsnSerAsnGlyLys                             880885890                                                                     TCTGTGTCATGGTCTGAACCAGGTGGAAGACAGGCGCCCAAGGGACAG3097                          SerValSerTrpSerGluProGlyGly ArgGlnAlaProLysGlyGln                             895900905                                                                     CACGTGTGGCAGCGCCTCTCTGTGCACGTGAAGACCAACGAGACGGCC3145                          HisValTrpGlnArgLeuSerValHisValL ysThrAsnGluThrAla                             910915920                                                                     TGTAACCAAACAGCCGTAATCAAACCCCTCACTAAAAGTTACCAAGGC3193                          CysAsnGlnThrAlaValIleLysProLeuThrLysSe rTyrGlnGly                             925930935                                                                     TCTGGCAAGAGCCTGACCTTTTCAGATGCCAGCACCAAGACCCTTTAC3241                          SerGlyLysSerLeuThrPheSerAspAlaSerThrLysThrLeuTyr                              940945950955                                                                  AATGTGGAAGAAGAGGACAATACCCCTTCTGCTCACTTCAGCCCTCCC3289                          AsnValGluGluGluAspAsnThrProSerAlaHisPheSerPro Pro                             960965970                                                                     AGCAGCCCTTCTATGGTGGTGCACCGACGCGGGCCACCCGTGGCCACC3337                          SerSerProSerMetValValHisArgArgGlyProProValAlaT hr                             975980985                                                                     ACACCACCTCTGCCACCCCATCTGACCGCAGAAGAGACCCCCCTGTTC3385                          ThrProProLeuProProHisLeuThrAlaGluGluThrProLeuPhe                               9909951000                                                                   CTGGCTGATTCCGTCATCCCCAAGGGCTTGCCTCCTCCTCTCCCGCAG3433                          LeuAlaAspSerValIleProLysGlyLeuProProProLeuProGln                              1005 10101015                                                                 CAGCAGCCACAGCAGCCGCCCCCTCAGCAGCCCCCGCAGCAGCCCAAG3481                          GlnGlnProGlnGlnProProProGlnGlnProProGlnGlnProLys                              10201 02510301035                                                             TCCCTGATGGACCAGCTGCAAGGCGTAGTCACCAACTTCGGTTCGGGG3529                          SerLeuMetAspGlnLeuGlnGlyValValThrAsnPheGlySerGly                              1 04010451050                                                                 ATTCCAGATTTCCATGCGGTGCTGGCAGGCCCGGGGACACCAGGAAAC3577                          IleProAspPheHisAlaValLeuAlaGlyProGlyThrProGlyAsn                              1055 10601065                                                                 AGCCTGCGCTCTCTGTACCCGCCCCCGCCTCCGCCGCAACACCTGCAG3625                          SerLeuArgSerLeuTyrProProProProProProGlnHisLeuGln                              1070 10751080                                                                 ATGCTGCCCCTGCACCTGAGCACCTTCCAGGAGGAGTCCATCTCCCCT3673                          MetLeuProLeuHisLeuSerThrPheGlnGluGluSerIleSerPro                              10851090 1095                                                                 CCTGGGGAGGACATCGATGATGACAGTGAGAGATTCAAGCTCCTGCAG3721                          ProGlyGluAspIleAspAspAspSerGluArgPheLysLeuLeuGln                              110011051 1101115                                                             GAGTTCGTGTACGAGCGCGAAGGGAACACCGAAGAAGATGAATTGGAA3769                          GluPheValTyrGluArgGluGlyAsnThrGluGluAspGluLeuGlu                              11201 1251130                                                                 GAGGAGGAGGACCTGCCCACAGCCAGCAAGCTGACCCCTGAGGATTCT3817                          GluGluGluAspLeuProThrAlaSerLysLeuThrProGluAspSer                              11351140 1145                                                                 CCTGCCCTGACGCCTCCTTCTCCTTTCCGAGATTCCGTGGCCTCTGGC3865                          ProAlaLeuThrProProSerProPheArgAspSerValAlaSerGly                              11501155 1160                                                                 AGCTCAGTGCCCAGTTCCCCCGTATCTGAGTCGGTCCTCTGCACCCCT3913                          SerSerValProSerSerProValSerGluSerValLeuCysThrPro                              116511701175                                                                  CCAAATGTAACCTACGCCTCTGTCATTCTGAGGGACTACAAGCAAAGC3961                          ProAsnValThrTyrAlaSerValIleLeuArgAspTyrLysGlnSer                              1180118511901 195                                                             TCTTCCACCCTGTAGTGTGTGTGTGTGTGTGGGGGCGGGGGGAGTGCGCATG4013                      SerSerThrLeu                                                                  GAGAAGCCAGAGATGCCAAGGAGTGTCAACCCTTCCAGAAATGTGTAGAAAGCAGGGTGA4073              GGGATGGGGATGGAGGACCACGGTCTGCA GGGAAGAAAAAAAAAATGCTGCGGCTGCCTT4133             AAAGAAGGAGAGGGACGATGCCAACTGAACAGTGGTCCTGGCCAGGATTGTGACTCTTGA4193              ATTATTCAAAAACCTTCTCTAGAAAGAAAGGGAATTATGACAAAGCACAATTCCATATGG4253              TATGTAACTTT TATCGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA4300                          (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1199 amino acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetValArgLeuLeuLeuIlePh ePheProMetIlePheLeuGluMet                             151015                                                                        SerIleLeuProArgMetProAspArgLysValLeuLeuAlaGlyAla                              2025 30                                                                       SerSerGlnArgSerValAlaArgMetAspGlyAspValIleIleGly                              354045                                                                        AlaLeuPheSerValHisHisGlnProProAlaGluLysValProG lu                             505560                                                                        ArgLysCysGlyGluIleArgGluGlnTyrGlyIleGlnArgValGlu                              65707580                                                                      AlaMetPheHis ThrLeuAspLysIleAsnAlaAspProValLeuLeu                             859095                                                                        ProAsnIleThrLeuGlySerGluIleArgAspSerCysTrpHisSer                              100 105110                                                                    SerValAlaLeuGluGlnSerIleGluPheIleArgAspSerLeuIle                              115120125                                                                     SerIleArgAspGluLysAspGlyLeuAsnArgCy sLeuProAspGly                             130135140                                                                     GlnThrLeuProProGlyArgThrLysLysProIleAlaGlyValIle                              145150155160                                                                   GlyProGlySerSerSerValAlaIleGlnValGlnAsnLeuLeuGln                             165170175                                                                     LeuPheAspIleProGlnIleAlaTyrSerAlaThrSerIleAspLeu                               180185190                                                                    SerAspLysThrLeuTyrLysTyrPheLeuArgValValProSerAsp                              195200205                                                                     ThrLeuGlnAlaArgAlaMetLeu AspIleValLysArgTyrAsnTrp                             210215220                                                                     ThrTyrValSerAlaValHisThrGluGlyAsnTyrGlyGluSerGly                              225230235 240                                                                 MetAspAlaPheLysGluLeuAlaAlaGlnGluGlyLeuCysIleAla                              245250255                                                                     HisSerAspLysIleTyrSerAsnAlaGlyGluLysSerPheAs pArg                             260265270                                                                     LeuLeuArgLysLeuArgGluArgLeuProLysAlaArgValValVal                              275280285                                                                     CysPheCysGlu GlyMetThrValArgGlyLeuLeuSerAlaMetArg                             290295300                                                                     ArgLeuGlyValValGlyGluPheSerLeuIleGlySerAspGlyTrp                              305310 315320                                                                 AlaAspArgAspGluValIleGluGlyTyrGluValGluAlaAsnGly                              325330335                                                                     GlyIleThrIleLysLeuGlnSerProGluVal ArgSerPheAspAsp                             340345350                                                                     TyrPheLeuLysLeuArgLeuAspThrAsnThrArgAsnProTrpPhe                              355360365                                                                     P roGluPheTrpGlnHisArgPheGlnCysArgLeuProGlyHisLeu                             370375380                                                                     LeuGluAsnProAsnPheLysLysValCysThrGlyAsnGluSerLeu                              3853 90395400                                                                 GluGluAsnTyrValGlnAspSerLysMetGlyPheValIleAsnAla                              405410415                                                                     IleTyrAlaMetAlaHisGly LeuGlnAsnMetHisHisAlaLeuCys                             420425430                                                                     ProGlyHisValGlyLeuCysAspAlaMetLysProIleAspGlyArg                              435440 445                                                                    LysLeuLeuAspPheLeuIleLysSerSerPheValGlyValSerGly                              450455460                                                                     GluGluValTrpPheAspGluLysGlyAspAlaProGlyArgTyrAsp                              465 470475480                                                                 IleMetAsnLeuGlnTyrThrGluAlaAsnArgTyrAspTyrValHis                              485490495                                                                     ValGlyThrT rpHisGluGlyValLeuAsnIleAspAspTyrLysIle                             500505510                                                                     GlnMetAsnLysSerGlyMetValArgSerValCysSerGluProCys                              515 520525                                                                    LeuLysGlyGlnIleLysValIleArgLysGlyGluValSerCysCys                              530535540                                                                     TrpIleCysThrAlaCysLysGluAsnGluPheValGlnAsp GluPhe                             545550555560                                                                  ThrCysArgAlaCysAspLeuGlyTrpTrpProAsnAlaGluLeuThr                              565570575                                                                     GlyCysGluProIleProValArgTyrLeuGluTrpSerAspIleGlu                              580585590                                                                     SerIleIleAlaIleAlaPheSerCysLeuGlyIleLeuValThrLeu                              59 5600605                                                                    PheValThrLeuIlePheValLeuTyrArgAspThrProValValLys                              610615620                                                                     SerSerSerArgGluLeuCysTyrIleIleL euAlaGlyIlePheLeu                             625630635640                                                                  GlyTyrValCysProPheThrLeuIleAlaLysProThrThrThrSer                              645650 655                                                                    CysTyrLeuGlnArgLeuLeuValGlyLeuSerSerAlaMetCysTyr                              660665670                                                                     SerAlaLeuValThrLysThrAsnArgIleAlaArgIleLeuAla Gly                             675680685                                                                     SerLysLysLysIleCysThrArgLysProArgPheMetSerAlaTrp                              690695700                                                                     AlaGlnValIleIleAlaSe rIleLeuIleSerValGlnLeuThrLeu                             705710715720                                                                  ValValThrLeuIleIleMetGluProProMetProIleLeuSerTyr                              725 730735                                                                    ProSerIleLysGluValTyrLeuIleCysAsnThrSerAsnLeuGly                              740745750                                                                     ValValAlaProValGlyTyrAsnGlyLeuLeuI leMetSerCysThr                             755760765                                                                     TyrTyrAlaPheLysThrArgAsnValProAlaAsnPheAsnGluAla                              770775780                                                                     LysTyrIle AlaPheThrMetTyrThrThrCysIleIleTrpLeuAla                             785790795800                                                                  PheValProIleTyrPheGlySerAsnTyrLysIleIleThrThrCys                               805810815                                                                    PheAlaValSerLeuSerValThrValAlaLeuGlyCysMetPheThr                              820825830                                                                     ProLysMetTyrIleIleIleAl aLysProGluArgAsnValArgSer                             835840845                                                                     AlaPheThrThrSerAspValValArgMetHisValGlyAspGlyLys                              85085586 0                                                                    LeuProCysArgSerAsnThrPheLeuAsnIlePheArgArgLysLys                              865870875880                                                                  ProGlyAlaGlyAsnAlaAsnSerAsnGlyLysSerValSerTrpSer                               885890895                                                                    GluProGlyGlyArgGlnAlaProLysGlyGlnHisValTrpGlnArg                              900905910                                                                     LeuSerValHis ValLysThrAsnGluThrAlaCysAsnGlnThrAla                             915920925                                                                     ValIleLysProLeuThrLysSerTyrGlnGlySerGlyLysSerLeu                              930935 940                                                                    ThrPheSerAspAlaSerThrLysThrLeuTyrAsnValGluGluGlu                              945950955960                                                                  AspAsnThrProSerAlaHisPheSerProProSerSe rProSerMet                             965970975                                                                     ValValHisArgArgGlyProProValAlaThrThrProProLeuPro                              980985990                                                                      ProHisLeuThrAlaGluGluThrProLeuPheLeuAlaAspSerVal                             99510001005                                                                   IleProLysGlyLeuProProProLeuProGlnGlnGlnProGlnGln                              1010 10151020                                                                 ProProProGlnGlnProProGlnGlnProLysSerLeuMetAspGln                              1025103010351040                                                              LeuGlnGlyValValThrAsnPhe GlySerGlyIleProAspPheHis                             104510501055                                                                  AlaValLeuAlaGlyProGlyThrProGlyAsnSerLeuArgSerLeu                              10601065 1070                                                                 TyrProProProProProProGlnHisLeuGlnMetLeuProLeuHis                              107510801085                                                                  LeuSerThrPheGlnGluGluSerIleSerProProGlyGluAsp Ile                             109010951100                                                                  AspAspAspSerGluArgPheLysLeuLeuGlnGluPheValTyrGlu                              1105111011151120                                                              ArgGluGly AsnThrGluGluAspGluLeuGluGluGluGluAspLeu                             112511301135                                                                  ProThrAlaSerLysLeuThrProGluAspSerProAlaLeuThrPro                              1140 11451150                                                                 ProSerProPheArgAspSerValAlaSerGlySerSerValProSer                              115511601165                                                                  SerProValSerGluSerValLeuCysThr ProProAsnValThrTyr                             117011751180                                                                  AlaSerValIleLeuArgAspTyrLysGlnSerSerSerThrLeu                                 118511901195                                                                  (2) INFORMATION FOR SEQ ID NO:3:                                               (i) SEQUENCE CHARACTERISTICS:                                                (A) LENGTH: 35 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: ZC775                                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GCTAGCATAACCCCTTGGGGCCTCTAAACGGGTCT35                                         (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: ZC776                                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       CTCAAGACCCGTTTAGAGGCCCCAAGGGGTTATGCTAGCTGCA 43                                (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 40 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: ZC777                                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       TGAGGGGTTTTTTGCTGAAAGGAGGAACTA TGCGGCCGCA40                                   (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 40 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: ZC778                                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       AGCTTGCGGCCGCATAGT TCCTCCTTTCAGCAAAAAACCC40                                   (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: ZC1751                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       AATTCT GTGCTCTGTCAAG19                                                        (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: ZC1752                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       GATCCTTGACAGAG CACAG19                                                        (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: ZC2063                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       GATCCAAACTAGTAAAAGAGCT 22                                                     (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: ZC2064                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      CTTTTACTAGTTTG14                                                               (2) INFORMATION FOR SEQ ID NO:11:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: ZC2938                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      GACAGAGCACAGATTCACTAGTGAGCTCTTTTTTTTTTT TTTT43                                (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: ZC3015                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      TTCCATGGCACCGTCAAGGCT 21                                                      (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: ZC3016                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      AGTGATGGCATGGACTGTGGT21                                                       (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: ZC3652                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      ACATGCACCATGCTCTGTGT20                                                        (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: ZC3654                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      AGTGATGGCATGGACTGTGGT21                                                   

What is claimed is:
 1. An isolated and purified polynucleotide moleculewhich hybridizes at high stringency to an oligonucleotide of 25 or morecontiguous nucleotides of SEQ ID NO: 1 and which codes for:a) amammalian G protein coupled glutamate receptor; or b) a ligand bindingdomain or a transmembrane domain of a mammalian G protein coupledglutamate receptor.
 2. The polynucleotide of claim 1, which is an RNAantisense sequence.
 3. The polynucleotide of claim 1, which encodes apolypeptide displaying mammalian G protein coupled glutamate receptoractivity.
 4. The polynucleotide of claim 1, which encodes a mammalian Gprotein coupled glutamate receptor having the amino acid sequence of SEQID NO:
 2. 5. A probe which comprises an oligonucleotide of 25 or morecontiguous nucleotides of SEQ ID NO: 1 capable of specificallyhybridizing with a gene which encodes a mammalian G protein coupledglutamate receptor, or allelic and species variants thereof.
 6. Theprobe of claim 5, which comprises from about 40 to about 60 nucleotidesin length.
 7. The probe of claim 6, which is labeled to provide adetectable signal.
 8. A DNA construct comprising the following operablylinked elements:a transcriptional promoter; a DNA sequence whichhybridizes at high stringency to an oligonucleotide of 25 or morecontiguous nucleotides of SEQ ID NO: 1 and which encodes (a) a mammalianG protein coupled glutamate receptor; or (b) a ligand binding domain ora transmembrane domain of a mammalian G protein coupled glutamatereceptor; and a transcriptional terminator.
 9. The DNA construct ofclaim 8, wherein the DNA sequence encodes a mammalian G protein coupledglutamate receptor having the amino acid sequence of SEQ ID NO:
 2. 10. Acultured eukaryotic cell transformed or transfected with a DNA constructwhich comprises the following operably linked elements:a transcriptionalpromoter; a DNA sequence which hybridizes at high stringency to anoligonucleotide of 25 or more contiguous nucleotides of SEQ ID NO: 1 andwhich encodes (a) a mammalian G protein coupled glutamate receptor; or(b) a ligand binding domain or a transmembrane domain of a mammalian Gprotein coupled glutamate receptor; and a transcriptional terminator.11. The eukaryotic cell of claim 10, which is a mammalian cell.
 12. Theeukaryotic cell of claim 10, which does not express endogenous G proteincoupled glutamate receptors.
 13. The DNA construct of claim 10, whereinthe DNA sequence encodes a mammalian G protein coupled glutamatereceptor having the amino acid sequence of SEQ ID NO:
 2. 14. A methodfor producing a mammalian G protein coupled glutamate receptor havingthe amino acid sequence of SEQ ID NO: 2, which comprises:growingeukaryotic cells transformed or transfected with a DNA construct whichcomprises a DNA sequence of SEQ ID NO: 1 coding for the expression ofthe G protein coupled glutamate receptor, and isolating the receptorfrom the cells.
 15. The method of claim 14, wherein the cells arecultured mammalian cells.
 16. The method of claim 14, wherein theglutamate receptor is isolated by immunoaffinity purification.
 17. Themethod of claim 14, wherein the G protein coupled glutamate receptor isnot coupled to G protein in the eukaryotic cells.